Compositions and Methods for TTR Gene Editing and Treating ATTR Amyloidosis

ABSTRACT

Compositions and methods for editing, e.g., introducing double-stranded breaks, within the TTR gene are provided. Compositions and methods for treating subjects having amyloidosis associated with transthyretin (ATTR), are provided.

This application is a Continuation of International Application No.PCT/US2018/053382, which was filed on Sep. 28, 2018, which claims thebenefit of priority to U.S. Provisional Application No. 62/556,236,which was filed on Sep. 29, 2017, and U.S. Provisional Application No.62/671,902, which was filed on May 15, 2018, the contents of each ofwhich are incorporated by reference in their entirety.

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 23, 2020, isnamed 2020-03-23_01155-0013-US_ST25.txt and is 417,533 bytes in size.

Transthyretin (TTR) is a protein produced by the TTR gene that normallyfunctions to transport retinol and thyroxine throughout the body. TTR ispredominantly synthesized in the liver, with small fractions beingproduced in the choroid plexus and retina. TTR normally circulates as asoluble tetrameric protein in the blood.

Pathogenic variants of TTR, which may disrupt tetramer stability, can beencoded by mutant alleles of the TTR gene. Mutant TTR may result inmisfolded TTR, which may generate amyloids (i.e., aggregates ofmisfolded TTR protein). In some cases, pathogenic variants of TTR canlead to amyloidosis, or disease resulting from build-up of amyloids. Forexample, misfolded TTR monomers can polymerize into amyloid fibrilswithin tissues, such as the peripheral nerves, heart, andgastrointestinal tract. Amyloid plaques can also comprise wild-type TTRthat has deposited on misfolded TTR.

Misfolding and deposition of wild-type TTR has also been observed inmales aged 60 or more and is associated with heart rhythm problems,heart failure, and carpal tunnel.

Amyloidosis characterized by deposition of TTR may be referred to as“ATTR,” “TTR-related amyloidosis,” “TTR amyloidosis,” or “ATTRamyloidosis,” “ATTR familial amyloidosis” (when associated with agenetic mutation in a family), or “ATTRwt” or “wild-type ATTR” (whenarising from misfolding and deposition of wild-type TTR).

ATTR can present with a wide spectrum of symptoms, and patients withdifferent classes of ATTR may have different characteristics andprognoses. Some classes of ATTR include familial amyloid polyneuropathy(FAP), familial amyloid cardiomyopathy (FAC), and wild-type TTRamyloidosis (wt-TTR amyloidosis). FAP commonly presents withsensorimotor neuropathy, while FAC and wt-TTR amyloidosis commonlypresent with congestive heart failure. FAP and FAC are usuallyassociated with a genetic mutation in the TTR gene, and more than 100different mutations in the TTR gene have been associated with ATTR. Incontrast, wt-TTR amyloidosis is associated with aging and not with agenetic mutation in TTR. It is estimated that approximately 50,000patients worldwide may be affected by FAP and FAC.

While more than 100 mutations in TTR are associated with ATTR, certainmutations have been more closely associated with neuropathy and/orcardiomyopathy. For example, mutations at T60 of TTR are associated withboth cardiomyopathy and neuropathy; mutations at V30 are more associatedwith neuropathy; and mutations at V122 are more associated withcardiomyopathy.

A range of treatment approaches have been studied for treatment of ATTR,but there are no approved drugs that stop disease progression andimprove quality of life. While liver transplant has been studied fortreatment of ATTR, its use is declining as it involves significant riskand disease progression sometimes continues after transplantation. Smallmolecule stabilizers, such as diflunisal and tafamidis, appear to slowATTR progression, but these agents do not halt disease progression.

Approaches using small interfering RNA (siRNA) knockdown, antisenseknockdown, or a monoclonal antibody targeting amyloid fibrils fordestruction are also currently being investigated, but while results onshort-term suppression of TTR expression show encouraging preliminarydata, a need exists for treatments that can produce long-lastingsuppression of TTR.

Accordingly, the following embodiments are provided. In someembodiments, the present invention provides compositions and methodsusing a guide RNA with an RNA-guided DNA binding agent such as theCRISPR/Cas system to substantially reduce or knockout expression of theTTR gene, thereby substantially reducing or eliminating the productionof TTR protein associated with ATTR. The substantial reduction orelimination of the production of TTR protein associated with ATTRthrough alteration of the TTR gene can be a long-term reduction orelimination.

SUMMARY

Embodiment 1 is a method of inducing a double-stranded break (DSB)within the TTR gene, comprising delivering a composition to a cell,wherein the composition comprises

a. a guide RNA comprising a guide sequence selected from SEQ ID NOs:5-82;b. a guide RNA comprising at least 17, 18, 19, or 20 contiguousnucleotides of a sequence selected from SEQ ID NOs: 5-82; orc. a guide RNA comprising a guide sequence that is at least 99%, 98%,97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to a sequenceselected from SEQ ID NOs: 5-82.

Embodiment 2 is a method of modifying the TTR gene comprising deliveringa composition to a cell, wherein the composition comprises (i) anRNA-guided DNA binding agent or a nucleic acid encoding an RNA-guidedDNA binding agent and (ii) a guide RNA comprising:

a. a guide sequence selected from SEQ ID NOs: 5-82;b. at least 17, 18, 19, or 20 contiguous nucleotides of a sequenceselected from SEQ ID NOs: 5-82; orc. a guide sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%,92%, 91%, or 90% identical to a sequence selected from SEQ ID NOs: 5-82.

Embodiment 3 is a method of treating amyloidosis associated with TTR(ATTR), comprising administering a composition to a subject in needthereof, wherein the composition comprises (i) an RNA-guided DNA bindingagent or a nucleic acid encoding an RNA-guided DNA binding agent and(ii) a guide RNA comprising:

a. a guide sequence selected from SEQ ID NOs: 5-82;b. at least 17, 18, 19, or 20 contiguous nucleotides of a sequenceselected from SEQ ID NOs: 5-82; orc. a guide sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%,92%, 91%, or 90% identical to a sequence selected from SEQ ID NOs: 5-82,thereby treating ATTR.

Embodiment 4 is a method of reducing TTR serum concentration, comprisingadministering a composition to a subject in need thereof, wherein thecomposition comprises (i) an RNA-guided DNA binding agent or a nucleicacid encoding an RNA-guided DNA binding agent and (ii) a guide RNAcomprising:

a. a guide sequence selected from SEQ ID NOs: 5-82;b. at least 17, 18, 19, or 20 contiguous nucleotides of a sequenceselected from SEQ ID NOs: 5-82; orc. a guide sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%,92%, 91%, or 90% identical to a sequence selected from SEQ ID NOs: 5-82,thereby reducing TTR serum concentration.

Embodiment 5 is a method for reducing or preventing the accumulation ofamyloids or amyloid fibrils comprising TTR in a subject, comprisingadministering a composition to a subject in need thereof, wherein thecomposition comprises (i) an RNA-guided DNA binding agent or a nucleicacid encoding an RNA-guided DNA binding agent and (ii) a guide RNAcomprising:

a. a guide sequence selected from SEQ ID NOs: 5-82;b. at least 17, 18, 19, or 20 contiguous nucleotides of a sequenceselected from SEQ ID NOs: 5-82; orc. a guide sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%,92%, 91%, or 90% identical to a sequence selected from SEQ ID NOs: 5-82,thereby reducing accumulation of amyloids or amyloid fibrils.

Embodiment 6 is a composition comprising a guide RNA comprising:

a. a guide sequence selected from SEQ ID NOs: 5-82;b. at least 17, 18, 19, or 20 contiguous nucleotides of a sequenceselected from SEQ ID NOs: 5-82; orc. a guide sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%,92%, 91%, or 90% identical to a sequence selected from SEQ ID NOs: 5-82.

Embodiment 7 is a composition comprising a vector encoding a guide RNA,wherein the guide RNA comprises:

a. a guide sequence selected from SEQ ID NOs: 5-82;b. at least 17, 18, 19, or 20 contiguous nucleotides of a sequenceselected from SEQ ID NOs: 5-82; orc. a guide sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%,92%, 91%, or 90% identical to a sequence selected from SEQ ID NOs: 5-82.

Embodiment 8 is the composition of embodiment 6 or 7, for use ininducing a double-stranded break (DSB) within the TTR gene in a cell orsubject.

Embodiment 9 is the composition of embodiment 6 or 7, for use inmodifying the TTR gene in a cell or subject.

Embodiment 10 is the composition of embodiment 6 or 7, for use intreating amyloidosis associated with TTR (ATTR) in a subject.

Embodiment 11 is the composition of embodiment 6 or 7, for use inreducing TTR serum concentration in a subject.

Embodiment 12 is the composition of embodiment 6 or 7, for use inreducing or preventing the accumulation of amyloids or amyloid fibrilsin a subject.

Embodiment 13 is the method of any one of embodiments 1-5 or thecomposition for use of any one of embodiments 8-12, wherein thecomposition reduces serum TTR levels.

Embodiment 14 is the method or composition for use of embodiment 13,wherein the serum TTR levels are reduced by at least 50% as compared toserum TTR levels before administration of the composition.

Embodiment 15 is the method or composition for use of embodiment 13,wherein the serum TTR levels are reduced by 50-60%, 60-70%, 70-80%,80-90%, 90-95%, 95-98%, 98-99%, or 99-100% as compared to serum TTRlevels before administration of the composition.

Embodiment 16 is the method or composition for use of any one ofembodiments 1-5 or 8-15, wherein the composition results in editing ofthe TTR gene.

Embodiment 17 is the method or composition for use of embodiment 16,wherein the editing is calculated as a percentage of the population thatis edited (percent editing).

Embodiment 18 is the method or composition for use of embodiment 17,wherein the percent editing is between 30 and 99% of the population.

Embodiment 19 is the method or composition for use of embodiment 17,wherein the percent editing is between 30 and 35%, 35 and 40%, 40 and45%, 45 and 50%, 50 and 55%, 55 and 60%, 60 and 65%, 65 and 70%, 70 and75%, 75 and 80%, 80 and 85%, 85 and 90%, 90 and 95%, or 95 and 99% ofthe population.

Embodiment 20 is the method of any one of embodiments 1-5 or thecomposition for use of any one of embodiments 8-19, wherein thecomposition reduces amyloid deposition in at least one tissue.

Embodiment 21 is the method or composition for use of embodiment 20,wherein the at least one tissue comprises one or more of stomach, colon,sciatic nerve, or dorsal root ganglion.

Embodiment 22 is the method or composition for use of embodiment 20 or21, wherein amyloid deposition is measured 8 weeks after administrationof the composition.

Embodiment 23 is the method or composition for use of any one ofembodiments 20-22, wherein amyloid deposition is compared to a negativecontrol or a level measured before administration of the composition.

Embodiment 24 is the method or composition for use of any one ofembodiments 20-23, wherein amyloid deposition is measured in a biopsysample and/or by immunostaining.

Embodiment 25 is the method or composition for use of any one ofembodiments 20-24, wherein amyloid deposition is reduced by between 30and 35%, 35 and 40%, 40 and 45%, 45 and 50%, 50 and 55%, 55 and 60%, 60and 65%, 65 and 70%, 70 and 75%, 75 and 80%, 80 and 85%, 85 and 90%, 90and 95%, or 95 and 99% of the amyloid deposition seen in a negativecontrol.

Embodiment 26 is the method or composition for use of any one ofembodiments 20-25, wherein amyloid deposition is reduced by between 30and 35%, 35 and 40%, 40 and 45%, 45 and 50%, 50 and 55%, 55 and 60%, 60and 65%, 65 and 70%, 70 and 75%, 75 and 80%, 80 and 85%, 85 and 90%, 90and 95%, or 95 and 99% of the amyloid deposition seen beforeadministration of the composition.

Embodiment 27 is the method or composition for use of any one ofembodiments 1-5 or 8-26, wherein the composition is administered ordelivered at least two times.

Embodiment 28 is the method or composition for use of embodiment 27,wherein the composition is administered or delivered at least threetimes.

Embodiment 29 is the method or composition for use of embodiment 27,wherein the composition is administered or delivered at least fourtimes.

Embodiment 30 is the method or composition for use of embodiment 27,wherein the composition is administered or delivered up to five, six,seven, eight, nine, or ten times.

Embodiment 31 is the method or composition for use of any one ofembodiments 27-30, wherein the administration or delivery occurs at aninterval of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days.

Embodiment 32 is the method or composition for use of any one ofembodiments 27-30, wherein the administration or delivery occurs at aninterval of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 weeks.

Embodiment 33 is the method or composition for use of any one ofembodiments 27-30, wherein the administration or delivery occurs at aninterval of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 months.

Embodiment 34 is the method or composition of any one of the precedingembodiments, wherein the guide sequence is selected from SEQ ID NOs:5-82.

Embodiment 35 is the method or composition of any one of the precedingembodiments, wherein the guide RNA is at least partially complementaryto a target sequence present in the human TTR gene.

Embodiment 36 is the method or composition of embodiment 35, wherein thetarget sequence is in exon 1, 2, 3, or 4 of the human TTR gene.

Embodiment 37 is the method or composition of embodiment 35, wherein thetarget sequence is in exon 1 of the human TTR gene.

Embodiment 38 is the method or composition of embodiment 35, wherein thetarget sequence is in exon 2 of the human TTR gene.

Embodiment 39 is the method or composition of embodiment 35, wherein thetarget sequence is in exon 3 of the human TTR gene.

Embodiment 40 is the method or composition of embodiment 35, wherein thetarget sequence is in exon 4 of the human TTR gene.

Embodiment 41 is the method or composition of any one of embodiments1-40, wherein the guide sequence is complementary to a target sequencein the positive strand of TTR.

Embodiment 42 is the method or composition of any one of embodiments1-40, wherein the guide sequence is complementary to a target sequencein the negative strand of TTR.

Embodiment 43 is the method or composition of any one of embodiments1-40, wherein the first guide sequence is complementary to a firsttarget sequence in the positive strand of the TTR gene, and wherein thecomposition further comprises a second guide sequence that iscomplementary to a second target sequence in the negative strand of theTTR gene.

Embodiment 44 is the method or composition of any one of the precedingembodiments, wherein the guide RNA comprises a crRNA that comprises theguide sequence and further comprises a nucleotide sequence of SEQ ID NO:126, wherein the nucleotides of SEQ ID NO: 126 follow the guide sequenceat its 3′ end.

Embodiment 45 is the method or composition of any one of the precedingembodiments, wherein the guide RNA is a dual guide (dgRNA).

Embodiment 46 is the method or composition of embodiment 45, wherein thedual guide RNA comprises a crRNA comprising a nucleotide sequence of SEQID NO: 126, wherein the nucleotides of SEQ ID NO: 126 follow the guidesequence at its 3′ end, and a trRNA.

Embodiment 47 is the method or composition of any one of embodiments1-43, wherein the guide RNA is a single guide (sgRNA).

Embodiment 48 is the method or composition of embodiment 47, wherein thesgRNA comprises a guide sequence that has the pattern of SEQ ID NO: 3.

Embodiment 49 is the method or composition of embodiment 47, wherein thesgRNA comprises the sequence of SEQ ID NO: 3.

Embodiment 50 is the method or composition of embodiment 48 or 49,wherein each N in SEQ ID NO: 3 is any natural or non-natural nucleotide,wherein the N's form the guide sequence, and the guide sequence targetsCas9 to the TTR gene.

Embodiment 51 is the method or composition of any one of embodiments47-50, wherein the sgRNA comprises any one of the guide sequences of SEQID NOs: 5-82 and the nucleotides of SEQ ID NO: 126.

Embodiment 52 is the method or composition of any one of embodiments47-51, wherein the sgRNA comprises a guide sequence that is at least99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to asequence selected from SEQ ID Nos: 87-124.

Embodiment 53 is the method or composition of embodiment 47, wherein thesgRNA comprises a sequence selected from SEQ ID Nos: 87-124.

Embodiment 54 is the method or composition of any one of the precedingembodiments, wherein the guide RNA comprises at least one modification.

Embodiment 55 is the method or composition of embodiment 54, wherein theat least one modification includes a 2′-O-methyl (2′-O-Me) modifiednucleotide.

Embodiment 56 is the method or composition of embodiment 54 or 55,wherein the at least one modification includes a phosphorothioate (PS)bond between nucleotides.

Embodiment 57 is the method or composition of any one of embodiments54-56, wherein the at least one modification includes a 2′-fluoro (2′-F)modified nucleotide.

Embodiment 58 is the method or composition of any one of embodiments54-57, wherein the at least one modification includes a modification atone or more of the first five nucleotides at the 5′ end.

Embodiment 59 is the method or composition of any one of embodiments54-58, wherein the at least one modification includes a modification atone or more of the last five nucleotides at the 3′ end.

Embodiment 60 is the method or composition of any one of embodiments54-59, wherein the at least one modification includes PS bonds betweenthe first four nucleotides.

Embodiment 61 is the method or composition of any one of embodiments54-60, wherein the at least one modification includes PS bonds betweenthe last four nucleotides.

Embodiment 62 is the method or composition of any one of embodiments54-61, wherein the at least one modification includes 2′-O-Me modifiednucleotides at the first three nucleotides at the 5′ end.

Embodiment 63 is the method or composition of any one of embodiments54-62, wherein the at least one modification includes 2′-O-Me modifiednucleotides at the last three nucleotides at the 3′ end.

Embodiment 64 is the method or composition of any one of embodiments54-63, wherein the guide RNA comprises the modified nucleotides of SEQID NO: 3.

Embodiment 65 is the method or composition of any one of embodiments1-64, wherein the composition further comprises a pharmaceuticallyacceptable excipient.

Embodiment 66 is the method or composition of any one of embodiments1-65, wherein the guide RNA is associated with a lipid nanoparticle(LNP).

Embodiment 67 is the method or composition of embodiment 66, wherein theLNP comprises a CCD lipid.

Embodiment 68 is the method or composition of embodiment 67, wherein theCCD lipid is Lipid a or Lipid B.

Embodiment 69 is the method or composition of embodiment 66-68, whereinthe LNP comprises a neutral lipid.

Embodiment 70 is the method or composition of embodiment 69, wherein theneutral lipid is DSPC

Embodiment 71 is the method or composition of any one of embodiments66-70, wherein the LNP comprises a helper lipid.

Embodiment 72 is the method or composition of embodiment 71, wherein thehelper lipid is cholesterol.

Embodiment 73 is the method or composition of any one of embodiments66-72, wherein the LNP comprises a stealth lipid.

Embodiment 74 is the method or composition of embodiment 73, wherein thestealth lipid is PEG2k-DMG.

Embodiment 75 is the method or composition of any one of the precedingembodiments, wherein the composition further comprises an RNA-guided DNAbinding agent.

Embodiment 76 is the method or composition of any one of the precedingembodiments, wherein the composition further comprises an mRNA thatencodes an RNA-guided DNA binding agent.

Embodiment 77 is the method or composition of embodiment 75 or 76,wherein the RNA-guided DNA binding agent is a Cas cleavase.

Embodiment 78 is the method or composition of embodiment 77, wherein theRNA-guided DNA binding agent is Cas9.

Embodiment 79 is the method or composition of any one of embodiments75-78, wherein the RNA-guided DNA binding agent is modified.

Embodiment 80 is the method or composition of any one of embodiments75-79, wherein the RNA-guided DNA binding agent is a nickase.

Embodiment 81 is the method or composition of embodiment 79 or 80,wherein the modified RNA-guided DNA binding agent comprises a nuclearlocalization signal (NLS).

Embodiment 82 is the method or composition of any one of embodiments75-81, wherein the RNA-guided DNA binding agent is a Cas from a Type-IICRISPR/Cas system.

Embodiment 83 is the method or composition of any one of the precedingembodiments, wherein the composition is a pharmaceutical formulation andfurther comprises a pharmaceutically acceptable carrier.

Embodiment 84 is the method or composition for use of any one ofembodiments 1-5 or 8-83, wherein the composition reduces or preventsamyloids or amyloid fibrils comprising TTR.

Embodiment 85 is the method or composition for use of embodiment 84,wherein the amyloids or amyloid fibrils are in the nerves, heart, orgastrointestinal track.

Embodiment 86 is the method or composition for use of any one ofembodiments 1-5 or 8-83, wherein non-homologous ending joining (NHEJ)leads to a mutation during repair of a DSB in the TTR gene.

Embodiment 87 is the method or composition for use of embodiment 86,wherein NHEJ leads to a deletion or insertion of a nucleotide(s) duringrepair of a DSB in the TTR gene.

Embodiment 88 is the method or composition for use of embodiment 87,wherein the deletion or insertion of a nucleotide(s) induces a frameshift or nonsense mutation in the TTR gene.

Embodiment 89 is the method or composition for use of embodiment 87,wherein a frame shift or nonsense mutation is induced in the TTR gene ofat least 50% of liver cells.

Embodiment 90 is the method or composition for use of embodiment 89,wherein a frame shift or nonsense mutation is induced in the TTR gene of50%-60%, 60%-70%, 70% or 80%, 80%-90%, 90-95%, 95%-99%, or 99%-100% ofliver cells.

Embodiment 91 is the method or composition for use of any one ofembodiments 87-90, wherein a deletion or insertion of a nucleotide(s)occurs in the TTR gene at least 50-fold or more than in off-targetsites.

Embodiment 92 is the method or composition for use of embodiment 91,wherein the deletion or insertion of a nucleotide(s) occurs in the TTRgene 50-fold to 150-fold, 150-fold to 500-fold, 500-fold to 1500-fold,1500-fold to 5000-fold, 5000-fold to 15000-fold, 15000-fold to30000-fold, or 30000-fold to 60000-fold more than in off-target sites.

Embodiment 93 is the method or composition for use of any one ofembodiments 87-92, wherein the deletion or insertion of a nucleotide(s)occurs at less than or equal to 3, 2, 1, or 0 off-target site(s) inprimary human hepatocytes, optionally wherein the off-target site(s)does (do) not occur in a protein coding region in the genome of theprimary human hepatocytes.

Embodiment 94 is the method or composition for use of embodiment 93,wherein the deletion or insertion of a nucleotide(s) occurs at a numberof off-target sites in primary human hepatocytes that is less than thenumber of off-target sites at which a deletion or insertion of anucleotide(s) occurs in Cas9-overexpressing cells, optionally whereinthe off-target site(s) does (do) not occur in a protein coding region inthe genome of the primary human hepatocytes.

Embodiment 95 is the method or composition for use of embodiment 94,wherein the Cas9-overexpressing cells are HEK293 cells stably expressingCas9.

Embodiment 96 is the method or composition for use of any one ofembodiments 93-95, wherein the number of off-target sites in primaryhuman hepatocytes is determined by analyzing genomic DNA from primaryhuman hepatocytes transfected in vitro with Cas9 mRNA and the guide RNA,optionally wherein the off-target site(s) does (do) not occur in aprotein coding region in the genome of the primary human hepatocytes.

Embodiment 97 is the method or composition for use of any one ofembodiments 93-95, wherein the number of off-target sites in primaryhuman hepatocytes is determined by an oligonucleotide insertion assaycomprising analyzing genomic DNA from primary human hepatocytestransfected in vitro with Cas9 mRNA, the guide RNA, and a donoroligonucleotide, optionally wherein the off-target site(s) does (do) notoccur in a protein coding region in the genome of the primary humanhepatocytes.

Embodiment 98 is the method or composition of any one of embodiments1-43 or 47-97, wherein the sequence of the guide RNA is:

a) SEQ ID NO: 92 or 104;

b) SEQ ID NO: 87, 89, 96, or 113;

c) SEQ ID NO: 100, 102, 106, 111, or 112; or

d) SEQ ID NO: 88, 90, 91, 93, 94, 95, 97, 101, 103, 108, or 109,

optionally wherein the guide RNA does not produce indels at off-targetsite(s) that occur in a protein coding region in the genome of primaryhuman hepatocytes.

Embodiment 99 is the method or composition for use of any one ofembodiments 1-5 or 8-98, wherein administering the composition reduceslevels of TTR in the subject.

Embodiment 100 is the method or composition for use of embodiment 99,wherein the levels of TTR are reduced by at least 50%.

Embodiment 101 is the method or composition for use of embodiment 100,wherein the levels of TTR are reduced by 50%-60%, 60%-70%, 70% or 80%,80%-90%, 90-95%, 95%-99%, or 99%-100%.

Embodiment 102 is the method or composition for use of embodiment 100 or101, wherein the levels of TTR are measured in serum, plasma, blood,cerebral spinal fluid, or sputum.

Embodiment 103 is the method or composition for use of embodiment 100 or101, wherein the levels of TTR are measured in liver, choroid plexus,and/or retina.

Embodiment 104 is the method or composition for use of any one ofembodiments 99-103, wherein the levels of TTR are measured viaenzyme-linked immunosorbent assay (ELISA).

Embodiment 105 is the method or composition for use of any one ofembodiments 1-5 or 8-104, wherein the subject has ATTR.

Embodiment 106 is the method or composition for use of any one ofembodiments 1-5 or 8-105, wherein the subject is human.

Embodiment 107 is the method or composition for use of embodiment 105 or106, wherein the subject has ATTRwt.

Embodiment 108 is the method or composition for use of embodiment 105 or106, wherein the subject has hereditary ATTR.

Embodiment 109 is the method or composition for use of any one ofembodiments 1-5, 8-106, or 108, wherein the subject has a family historyof ATTR.

Embodiment 110 is the method or composition for use of any one ofembodiments 1-5, 8-106, or 108-109, wherein the subject has familialamyloid polyneuropathy.

Embodiment 111 is the method or composition for use of any one ofembodiments 1-5 or 8-110, wherein the subject has only or predominantlynerve symptoms of ATTR.

Embodiment 112 is the method or composition for use of any one ofembodiments 1-5 or 8-110, wherein the subject has familial amyloidcardiomyopathy.

Embodiment 113 is the method or composition for use of any one ofembodiments 1-5, 8-109, or 112, wherein the subject has only orpredominantly cardiac symptoms of ATTR.

Embodiment 114 is the method or composition for use of any one ofembodiments 1-5 or 8-113, wherein the subject expresses TTR having a V30mutation.

Embodiment 115 is the method or composition for use of embodiment 114,wherein the V30 mutation is V30A, V30G, V30L, or V30M.

Embodiment 116 is the method or composition for use of embodiment anyone of embodiments 1-5 or 8-113, wherein the subject expresses TTRhaving a T60 mutation.

Embodiment 117 is the method or composition for use of embodiment 116,wherein the T60 mutation is T60A.

Embodiment 118 is the method or composition for use of embodiment anyone of embodiments 1-5 or 8-113, wherein the subject expresses TTRhaving a V122 mutation.

Embodiment 119 is the method or composition for use of embodiment 118,wherein the V122 mutation is V122A, V122I, or V122(−).

Embodiment 120 is the method or composition for use of any one ofembodiments 1-5 or 8-119, wherein the subject expresses wild-type TTR.

Embodiment 121 is the method or composition for use of any one ofembodiments 1-5, 8-107, or 120, wherein the subject does not express TTRhaving a V30, T60, or V122 mutation.

Embodiment 122 is the method or composition for use of any one ofembodiments 1-5, 8-107, or 120-121, wherein the subject does not expressTTR having a pathological mutation.

Embodiment 123 is the method or composition for use of embodiment 121,wherein the subject is homozygous for wild-type TTR.

Embodiment 124 is the method or composition for use of any one ofembodiments 1-5 or 8-123, wherein after administration the subject hasan improvement, stabilization, or slowing of change in symptoms ofsensorimotor neuropathy.

Embodiment 125 is the method or composition for use of embodiment 124,wherein the improvement, stabilization, or slowing of change in sensoryneuropathy is measured using electromyogram, nerve conduction tests, orpatient-reported outcomes.

Embodiment 126 is the method or composition for use of any one ofembodiments 1-5 or 8-125, wherein the subject has an improvement,stabilization, or slowing of change in symptoms of congestive heartfailure.

Embodiment 127 is the method or composition for use of embodiment 126,wherein the improvement, stabilization, or slowing of change incongestive heart failure is measured using cardiac biomarker tests, lungfunction tests, chest x-rays, or electrocardiography.

Embodiment 128 is the method or composition for use of any one ofembodiments 1-5 or 8-127, wherein the composition or pharmaceuticalformulation is administered via a viral vector.

Embodiment 129 is the method or composition for use of any one ofembodiments 1-5 or 8-127, wherein the composition or pharmaceuticalformulation is administered via lipid nanoparticles.

Embodiment 130 is the method or composition for use of any one ofembodiments 1-5 or 8-129, wherein the subject is tested for specificmutations in the TTR gene before administering the composition orformulation.

Embodiment 131 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:5.

Embodiment 132 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:6.

Embodiment 133 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:7.

Embodiment 134 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:8.

Embodiment 135 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:9.

Embodiment 136 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:10.

Embodiment 137 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:11.

Embodiment 138 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:12.

Embodiment 139 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:13.

Embodiment 140 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:14.

Embodiment 141 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:15.

Embodiment 142 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:16.

Embodiment 143 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:17.

Embodiment 144 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:18.

Embodiment 145 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:19.

Embodiment 146 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:20.

Embodiment 147 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:21.

Embodiment 148 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:22.

Embodiment 149 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:23.

Embodiment 150 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:24.

Embodiment 151 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:25.

Embodiment 152 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:26.

Embodiment 153 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:27.

Embodiment 154 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:28.

Embodiment 155 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:29.

Embodiment 156 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:30.

Embodiment 157 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:31.

Embodiment 158 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:32.

Embodiment 159 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:33.

Embodiment 160 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:34.

Embodiment 161 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:35.

Embodiment 162 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:36.

Embodiment 163 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:37.

Embodiment 164 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:38.

Embodiment 165 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:39.

Embodiment 166 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:40.

Embodiment 167 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:41.

Embodiment 168 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:42.

Embodiment 169 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:43.

Embodiment 170 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:44.

Embodiment 171 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:45.

Embodiment 172 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:46.

Embodiment 173 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:47.

Embodiment 174 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:48.

Embodiment 175 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:49.

Embodiment 176 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:50.

Embodiment 177 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:51.

Embodiment 178 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:52.

Embodiment 179 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:53.

Embodiment 180 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:54.

Embodiment 181 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:55.

Embodiment 182 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:56.

Embodiment 183 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:57.

Embodiment 184 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:58.

Embodiment 185 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:59.

Embodiment 186 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:60.

Embodiment 187 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:61.

Embodiment 188 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:62.

Embodiment 189 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:63.

Embodiment 190 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:64.

Embodiment 191 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:65.

Embodiment 192 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:66.

Embodiment 193 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:67.

Embodiment 194 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:68.

Embodiment 195 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:69.

Embodiment 196 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:70.

Embodiment 197 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:71.

Embodiment 198 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:72.

Embodiment 199 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:73.

Embodiment 200 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:74.

Embodiment 201 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:75.

Embodiment 202 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:76.

Embodiment 203 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:77.

Embodiment 204 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:78.

Embodiment 205 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:79.

Embodiment 206 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:80.

Embodiment 207 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:81.

Embodiment 208 is the method or composition of any one of embodiments1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:82.

Embodiment 209 is a use of a composition or formulation of any ofembodiments 6-208 for the preparation of a medicament for treating ahuman subject having ATTR.

Also disclosed is the use of a composition or formulation of any of theforegoing embodiments for the preparation of a medicament for treating ahuman subject having ATTR. Also disclosed are any of the foregoingcompositions or formulations for use in treating ATTR or for use inmodifying (e.g., forming an indel in, or forming a frameshift ornonsense mutation in) a TTR gene.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic of chromosome 18 with the regions of the TTRgene that are targeted by the guide sequences provided in Table 1.

FIG. 2 shows off-target analysis in HEK293_Cas9 cells of certain dualguide RNAs targeting TTR. The on-target site is designated by a filledsquare for each dual guide RNA tested, whereas closed circles representa potential off-target site.

FIG. 3 shows off-target analysis in HEK_Cas9 cells of certain singleguide RNAs targeting TTR. The on-target site is designated by a filledsquare for each single guide RNA tested, whereas open circles representa potential off-target site.

FIG. 4 shows dose response curves of lipid nanoparticle formulated humanTTR specific sgRNAs on primary human hepatocytes.

FIG. 5 shows dose response curves of lipid nanoparticle formulated humanTTR specific sgRNAs on primary cyno hepatocytes.

FIG. 6 shows dose response curves of lipid nanoparticle formulated cynoTTR specific sgRNAs on primary cyno hepatocytes.

FIG. 7 shows percent editing (% edit) of TTR and reduction of secretedTTR following administration of the guide in HUH7 cells sequencesprovided on the x-axis. The values are normalized to the amount ofalpha-1-antitrypsin (AAT) protein.

FIG. 8 shows western blot analysis of intracellular TTR followingadministration of targeted guides (listed in Table 1) in HUH7 cells.

FIG. 9 shows percentage liver editing of TTR observed followingadministration of LNP formulations to mice with humanized (G481-G499) ormurine (G282) TTR. Note: the first three ‘0’s in each Guide ID isomitted from the Figure, for example “G481” is “G000481” in Tables 2 and3.

FIGS. 10A-B show serum TTR levels observed following the dosing regimensindicated on the horizontal axis as μg/ml (FIG. 10A) or percentage ofTSS control (FIG. 10B). MPK=mg/kg throughout.

FIGS. 11A-B show serum TTR levels observed following the dosing regimensindicated on the horizontal axis for 1 mg/kg (FIG. 11A) or 0.5 mg/kgdosages (FIG. 11B). Data for a single 2 mg/kg dose is included as theright column in both panels.

FIGS. 12A-B show percentage liver editing observed following the dosingregimens indicated on the horizontal axis for 1 mg/kg (FIG. 12A) or 0.5mg/kg dosages (FIG. 12B). FIG. 12C shows percentage liver editingobserved following a single dose at 0.5, 1, or 2 mg/kg.

FIG. 13 shows percent liver editing observed following administration ofLNP formulations to mice humanized with respect to the TTR gene. Note:the first three ‘0’s in each Guide ID is omitted from the Figure, forexample “G481” is “G000481” in Tables 2 and

FIGS. 14A-B show that there is correlation between liver editing (FIG.14A) and serum human TTR levels (FIG. 14B) following administration ofLNP formulations to mice humanized with respect to the TTR gene. Note:the first three ‘0’s in each Guide ID is omitted from the Figure, forexample “G481” is “G000481” in Tables 2 and 3.

FIGS. 15A-B show that there is a dose response with respect to percentediting (FIG. 15A) and serum TTR levels (FIG. 15B) in wild type micefollowing administration of LNP formulations comprising guide G502,which is cross homologous between mouse and cyno.

FIG. 16 shows dose response curves of lipid nanoparticle formulatedhuman TTR specific sgRNAs on primary cyno hepatocytes.

FIG. 17 shows dose response curves of lipid nanoparticle formulated cynoTTR specific sgRNAs on primary human hepatocytes.

FIG. 18 shows dose response curves of lipid nanoparticle formulated cynoTTR specific sgRNAs on primary cyno hepatocytes.

FIGS. 19A-D show serum TTR (% TSS; FIGS. 19A and 19C) and editingresults following dosing of LNP formulations at the indicated ratios andamounts (FIGS. 19B and 19D).

FIG. 20 shows off-target analysis of certain single guide RNAs inPrimary Human Hepatocytes (PHH) targeting TTR. In the graph, filledsquares represent the identification of the on-target cut site, whileopen circles represent the identification of potential off-target sites.

FIGS. 21A-B show percent editing on-target (ONT, FIG. 21A) and at twooff-target sites (OT2 and OT4) in primary human hepatocytes followingadministration of lipid nanoparticle formulated G000480. FIG. 21B is are-scaled version of the OT2, OT4, and negative control (Neg Cont) datain FIG. 21A.

FIGS. 22A-B show percent editing on-target (ONT, FIG. 22A) and at anoff-target site (OT4) in primary human hepatocytes followingadministration of lipid nanoparticle formulated G000486. FIG. 22B is are-scaled version of the OT4 and negative control (Neg Cont) data inFIG. 22A.

FIGS. 23A-B show percent editing (FIG. 23A) and number of insertion anddeletion events at the TTR locus (FIG. 23B). FIG. 23A shows percentediting at the TTR locus in control and treatment (dosed with lipidnanoparticle formulated TTR specific sgRNA) groups. FIG. 23B shows thenumber of insertion and deletion events at the TTR locus when editingwas observed in the treatment group of FIG. 23A.

FIGS. 24A-B show TTR levels in circulating serum (FIG. 24A) andcerebrospinal fluid (CSF) (FIG. 24B), respectively, in μg/mL for controland treatment (dosed with lipid nanoparticle formulated TTR specificsgRNA) groups. Treatment resulted in >99% knockdown of TTR levels inserum.

FIGS. 25A-D show immunohistochemistry images with staining for TTR instomach (FIG. 25A), colon (FIG. 25B), sciatic nerve (FIG. 25C), anddorsal root ganglion (DRG) (FIG. 25D) from control and treatment (dosedwith lipid nanoparticle formulated TTR specific sgRNA) mice. At right,bar graphs show reduction in TTR staining 8 weeks after treatment intreated mice as measured by percent occupied area for each tissue type.

FIGS. 26A-C show liver TTR editing (FIG. 26A) and serum TTR results (inμg/mL (FIG. 26B) and as percentage of TSS-treated control (FIG. 26C)),respectively, from humanized TTR mice dosed with LNP formulations acrossa range of doses with guides G000480, G000488, G000489 and G000502 andcontaining Cas9 mRNA (SEQ ID NO: 1) in a 1:1 ratio by weight to theguide.

FIGS. 27A-C show liver TTR editing (FIG. 27A) and serum TTR results (inμg/mL (FIG. 27B) and as percentage of TSS-treated control (FIG. 27C)),respectively, from humanized TTR mice dosed with LNP formulations acrossa range of doses with guides G000481, G000482, G000486 and G000499 andcontaining Cas9 mRNA (SEQ ID NO: 1) in a 1:1 ratio by weight to theguide.

FIGS. 28A-C show liver TTR editing (FIG. 28A) and serum TTR results (inμg/mL (FIG. 28B) and as percentage of TSS-treated control (FIG. 28C)),respectively, from humanized TTR mice dosed with LNP formulations acrossa range of doses with guides G000480, G000481, G000486, G000499 andG000502 and containing Cas9 mRNA (SEQ ID NO: 1) in a 1:2 ratio by weightto the guide.

FIG. 29 shows relative expression of TTR mRNA in primary humanhepatocytes (PHH) after treatment with LNPs comprising Cas9 mRNA and agRNA as indicated, as compared to negative (untreated) controls.

FIG. 30 shows relative expression of TTR mRNA in primary humanhepatocytes (PHH) after treatment with LNPs comprising Cas9 mRNA and agRNA as indicated, as compared to negative (untreated) controls.

DETAILED DESCRIPTION

Reference will now be made in detail to certain embodiments of theinvention, examples of which are illustrated in the accompanyingdrawings. While the invention will be described in conjunction with theillustrated embodiments, it will be understood that they are notintended to limit the invention to those embodiments. On the contrary,the invention is intended to cover all alternatives, modifications, andequivalents, which may be included within the invention as defined bythe appended claims.

Before describing the present teachings in detail, it is to beunderstood that the disclosure is not limited to specific compositionsor process steps, as such may vary. It should be noted that, as used inthis specification and the appended claims, the singular form “a”, “an”and “the” include plural references unless the context clearly dictatesotherwise. Thus, for example, reference to “a conjugate” includes aplurality of conjugates and reference to “a cell” includes a pluralityof cells and the like.

Numeric ranges are inclusive of the numbers defining the range. Measuredand measurable values are understood to be approximate, taking intoaccount significant digits and the error associated with themeasurement. Also, the use of “comprise”, “comprises”, “comprising”,“contain”, “contains”, “containing”, “include”, “includes”, and“including” are not intended to be limiting. It is to be understood thatboth the foregoing general description and detailed description areexemplary and explanatory only and are not restrictive of the teachings.

Unless specifically noted in the above specification, embodiments in thespecification that recite “comprising” various components are alsocontemplated as “consisting of” or “consisting essentially of” therecited components; embodiments in the specification that recite“consisting of” various components are also contemplated as “comprising”or “consisting essentially of” the recited components; and embodimentsin the specification that recite “consisting essentially of” variouscomponents are also contemplated as “consisting of” or “comprising” therecited components (this interchangeability does not apply to the use ofthese terms in the claims). The term “or” is used in an inclusive sense,i.e., equivalent to “and/or,” unless the context clearly indicatesotherwise.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the desired subject matter inany way. In the event that any material incorporated by referencecontradicts any term defined in this specification or any other expresscontent of this specification, this specification controls. While thepresent teachings are described in conjunction with various embodiments,it is not intended that the present teachings be limited to suchembodiments. On the contrary, the present teachings encompass variousalternatives, modifications, and equivalents, as will be appreciated bythose of skill in the art.

I. Definitions

Unless stated otherwise, the following terms and phrases as used hereinare intended to have the following meanings:

“Polynucleotide” and “nucleic acid” are used herein to refer to amultimeric compound comprising nucleosides or nucleoside analogs whichhave nitrogenous heterocyclic bases or base analogs linked togetheralong a backbone, including conventional RNA, DNA, mixed RNA-DNA, andpolymers that are analogs thereof. A nucleic acid “backbone” can be madeup of a variety of linkages, including one or more ofsugar-phosphodiester linkages, peptide-nucleic acid bonds (“peptidenucleic acids” or PNA; PCT No. WO 95/32305), phosphorothioate linkages,methylphosphonate linkages, or combinations thereof. Sugar moieties of anucleic acid can be ribose, deoxyribose, or similar compounds withsubstitutions, e.g., 2′ methoxy or 2′ halide substitutions. Nitrogenousbases can be conventional bases (A, G, C, T, U), analogs thereof (e.g.,modified uridines such as 5-methoxyuridine, pseudouridine, orN1-methylpseudouridine, or others); inosine; derivatives of purines orpyrimidines (e.g., N⁴-methyl deoxyguanosine, deaza- or aza-purines,deaza- or aza-pyrimidines, pyrimidine bases with substituent groups atthe 5 or 6 position (e.g., 5-methylcytosine), purine bases with asubstituent at the 2, 6, or 8 positions, 2-amino-6-methylaminopurine,O⁶-methylguanine, 4-thio-pyrimidines, 4-amino-pyrimidines,4-dimethylhydrazine-pyrimidines, and O⁴-alkyl-pyrimidines; U.S. Pat. No.5,378,825 and PCT No. WO 93/13121). For general discussion see TheBiochemistry of the Nucleic Acids 5-36, Adams et al., ed., 11^(th) ed.,1992). Nucleic acids can include one or more “abasic” residues where thebackbone includes no nitrogenous base for position(s) of the polymer(U.S. Pat. No. 5,585,481). A nucleic acid can comprise only conventionalRNA or DNA sugars, bases and linkages, or can include both conventionalcomponents and substitutions (e.g., conventional bases with 2′ methoxylinkages, or polymers containing both conventional bases and one or morebase analogs). Nucleic acid includes “locked nucleic acid” (LNA), ananalogue containing one or more LNA nucleotide monomers with a bicyclicfuranose unit locked in an RNA mimicking sugar conformation, whichenhance hybridization affinity toward complementary RNA and DNAsequences (Vester and Wengel, 2004, Biochemistry 43(42):13233-41). RNAand DNA have different sugar moieties and can differ by the presence ofuracil or analogs thereof in RNA and thymine or analogs thereof in DNA.

“Guide RNA”, “gRNA”, and “guide” are used herein interchangeably torefer to either a crRNA (also known as CRISPR RNA), or the combinationof a crRNA and a trRNA (also known as tracrRNA). The crRNA and trRNA maybe associated as a single RNA molecule (single guide RNA, sgRNA) or intwo separate RNA molecules (dual guide RNA, dgRNA). “Guide RNA” or“gRNA” refers to each type. The trRNA may be a naturally-occurringsequence, or a trRNA sequence with modifications or variations comparedto naturally-occurring sequences.

As used herein, a “guide sequence” refers to a sequence within a guideRNA that is complementary to a target sequence and functions to direct aguide RNA to a target sequence for binding or modification (e.g.,cleavage) by an RNA-guided DNA binding agent. A “guide sequence” mayalso be referred to as a “targeting sequence,” or a “spacer sequence.” Aguide sequence can be 20 base pairs in length, e.g., in the case ofStreptococcus pyogenes (i.e., Spy Cas9) and related Cas9homologs/orthologs. Shorter or longer sequences can also be used asguides, e.g., 15-, 16-, 17-, 18-, 19-, 21-, 22-, 23-, 24-, or25-nucleotides in length. For example, in some embodiments, the guidesequence comprises at least 17, 18, 19, or 20 contiguous nucleotides ofa sequence selected from SEQ ID NOs: 5-82. In some embodiments, thetarget sequence is in a gene or on a chromosome, for example, and iscomplementary to the guide sequence. In some embodiments, the degree ofcomplementarity or identity between a guide sequence and itscorresponding target sequence may be about 75%, 80%, 85%, 88%, 90%, 95%,96%, 97%, 98%, 99%, or 100%. For example, in some embodiments, the guidesequence comprises a sequence with about 75%, 80%, 85%, 88%, 90%, 95%,96%, 97%, 98%, 99%, or 100% identity to at least 17, 18, 19, or 20contiguous nucleotides of a sequence selected from SEQ ID NOs: 5-82. Insome embodiments, the guide sequence and the target region may be 100%complementary or identical. In other embodiments, the guide sequence andthe target region may contain at least one mismatch. For example, theguide sequence and the target sequence may contain 1, 2, 3, or 4mismatches, where the total length of the target sequence is at least17, 18, 19, 20 or more base pairs. In some embodiments, the guidesequence and the target region may contain 1-4 mismatches where theguide sequence comprises at least 17, 18, 19, 20 or more nucleotides. Insome embodiments, the guide sequence and the target region may contain1, 2, 3, or 4 mismatches where the guide sequence comprises 20nucleotides.

Target sequences for Cas proteins include both the positive and negativestrands of genomic DNA (i.e., the sequence given and the sequence'sreverse compliment), as a nucleic acid substrate for a Cas protein is adouble stranded nucleic acid. Accordingly, where a guide sequence issaid to be “complementary to a target sequence”, it is to be understoodthat the guide sequence may direct a guide RNA to bind to the reversecomplement of a target sequence. Thus, in some embodiments, where theguide sequence binds the reverse complement of a target sequence, theguide sequence is identical to certain nucleotides of the targetsequence (e.g., the target sequence not including the PAM) except forthe substitution of U for T in the guide sequence.

As used herein, an “RNA-guided DNA binding agent” means a polypeptide orcomplex of polypeptides having RNA and DNA binding activity, or aDNA-binding subunit of such a complex, wherein the DNA binding activityis sequence-specific and depends on the sequence of the RNA. ExemplaryRNA-guided DNA binding agents include Cas cleavases/nickases andinactivated forms thereof (“dCas DNA binding agents”). “Cas nuclease”,also called “Cas protein”, as used herein, encompasses Cas cleavases,Cas nickases, and dCas DNA binding agents. Cas cleavases/nickases anddCas DNA binding agents include a Csm or Cmr complex of a type IIICRISPR system, the Cas10, Csm1, or Cmr2 subunit thereof, a Cascadecomplex of a type I CRISPR system, the Cas3 subunit thereof, and Class 2Cas nucleases. As used herein, a “Class 2 Cas nuclease” is asingle-chain polypeptide with RNA-guided DNA binding activity, such as aCas9 nuclease or a Cpf1 nuclease. Class 2 Cas nucleases include Class 2Cas cleavases and Class 2 Cas nickases (e.g., H840A, D10A, or N863Avariants), which further have RNA-guided DNA cleavases or nickaseactivity, and Class 2 dCas DNA binding agents, in which cleavase/nickaseactivity is inactivated. Class 2 Cas nucleases include, for example,Cas9, Cpf1, C2c1, C2c2, C2c3, HF Cas9 (e.g., N497A, R661A, Q695A, Q926Avariants), HypaCas9 (e.g., N692A, M694A, Q695A, H698A variants),eSPCas9(1.0) (e.g, K810A, K1003A, R1060A variants), and eSPCas9(1.1)(e.g., K848A, K1003A, R1060A variants) proteins and modificationsthereof. Cpf1 protein, Zetsche et al., Cell, 163: 1-13 (2015), ishomologous to Cas9, and contains a RuvC-like nuclease domain. Cpf1sequences of Zetsche are incorporated by reference in their entirety.See, e.g., Zetsche, Tables S1 and S3. “Cas9” encompasses Spy Cas9, thevariants of Cas9 listed herein, and equivalents thereof. See, e.g.,Makarova et al., Nat Rev Microbiol, 13(11): 722-36 (2015); Shmakov etal., Molecular Cell, 60:385-397 (2015).

“Modified uridine” is used herein to refer to a nucleoside other thanthymidine with the same hydrogen bond acceptors as uridine and one ormore structural differences from uridine. In some embodiments, amodified uridine is a substituted uridine, i.e., a uridine in which oneor more non-proton substituents (e.g., alkoxy, such as methoxy) takesthe place of a proton. In some embodiments, a modified uridine ispseudouridine. In some embodiments, a modified uridine is a substitutedpseudouridine, i.e., a pseudouridine in which one or more non-protonsubstituents (e.g., alkyl, such as methyl) takes the place of a proton.In some embodiments, a modified uridine is any of a substituted uridine,pseudouridine, or a substituted pseudouridine.

“Uridine position” as used herein refers to a position in apolynucleotide occupied by a uridine or a modified uridine. Thus, forexample, a polynucleotide in which “100% of the uridine positions aremodified uridines” contains a modified uridine at every position thatwould be a uridine in a conventional RNA (where all bases are standardA, U, C, or G bases) of the same sequence. Unless otherwise indicated, aU in a polynucleotide sequence of a sequence table or sequence listingin, or accompanying, this disclosure can be a uridine or a modifieduridine.

As used herein, a first sequence is considered to “comprise a sequencewith at least X % identity to” a second sequence if an alignment of thefirst sequence to the second sequence shows that X % or more of thepositions of the second sequence in its entirety are matched by thefirst sequence. For example, the sequence AAGA comprises a sequence with100% identity to the sequence AAG because an alignment would give 100%identity in that there are matches to all three positions of the secondsequence. The differences between RNA and DNA (generally the exchange ofuridine for thymidine or vice versa) and the presence of nucleosideanalogs such as modified uridines do not contribute to differences inidentity or complementarity among polynucleotides as long as therelevant nucleotides (such as thymidine, uridine, or modified uridine)have the same complement (e.g., adenosine for all of thymidine, uridine,or modified uridine; another example is cytosine and 5-methylcytosine,both of which have guanosine or modified guanosine as a complement).Thus, for example, the sequence 5′-AXG where X is any modified uridine,such as pseudouridine, N1-methyl pseudouridine, or 5-methoxyuridine, isconsidered 100% identical to AUG in that both are perfectlycomplementary to the same sequence (5′-CAU). Exemplary alignmentalgorithms are the Smith-Waterman and Needleman-Wunsch algorithms, whichare well-known in the art. One skilled in the art will understand whatchoice of algorithm and parameter settings are appropriate for a givenpair of sequences to be aligned; for sequences of generally similarlength and expected identity >50% for amino acids or >75% fornucleotides, the Needleman-Wunsch algorithm with default settings of theNeedleman-Wunsch algorithm interface provided by the EBI at thewww.ebi.ac.uk web server is generally appropriate.

“mRNA” is used herein to refer to a polynucleotide that is not DNA andcomprises an open reading frame that can be translated into apolypeptide (i.e., can serve as a substrate for translation by aribosome and amino-acylated tRNAs). mRNA can comprise a phosphate-sugarbackbone including ribose residues or analogs thereof, e.g., 2′-methoxyribose residues. In some embodiments, the sugars of an mRNAphosphate-sugar backbone consist essentially of ribose residues,2′-methoxy ribose residues, or a combination thereof. In general, mRNAsdo not contain a substantial quantity of thymidine residues (e.g., 0residues or fewer than 30, 20, 10, 5, 4, 3, or 2 thymidine residues; orless than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or0.1% thymidine content). An mRNA can contain modified uridines at someor all of its uridine positions.

As used herein, the “minimum uridine content” of a given open readingframe (ORF) is the uridine content of an ORF that (a) uses a minimaluridine codon at every position and (b) encodes the same amino acidsequence as the given ORF. The minimal uridine codon(s) for a givenamino acid is the codon(s) with the fewest uridines (usually 0 or 1except for a codon for phenylalanine, where the minimal uridine codonhas 2 uridines). Modified uridine residues are considered equivalent touridines for the purpose of evaluating minimum uridine content.

As used herein, the “minimum uridine dinucleotide content” of a givenopen reading frame (ORF) is the lowest possible uridine dinucleotide(UU) content of an ORF that (a) uses a minimal uridine codon (asdiscussed above) at every position and (b) encodes the same amino acidsequence as the given ORF. The uridine dinucleotide (UU) content can beexpressed in absolute terms as the enumeration of UU dinucleotides in anORF or on a rate basis as the percentage of positions occupied by theuridines of uridine dinucleotides (for example, AUUAU would have auridine dinucleotide content of 40% because 2 of 5 positions areoccupied by the uridines of a uridine dinucleotide). Modified uridineresidues are considered equivalent to uridines for the purpose ofevaluating minimum uridine dinucleotide content.

As used herein, “TTR” refers to transthyretin, which is the gene productof a TTR gene.

As used herein, “amyloid” refers to abnormal aggregates of proteins orpeptides that are normally soluble. Amyloids are insoluble, and amyloidscan create proteinaceous deposits in organs and tissues. Proteins orpeptides in amyloids may be misfolded into a form that allows manycopies of the protein to stick together to form fibrils. While someforms of amyloid may have normal functions in the human body, “amyloids”as used herein refers to abnormal or pathologic aggregates of protein.Amyloids may comprise a single protein or peptide, such as TTR, or theymay comprise multiple proteins or peptides, such as TTR and additionalproteins.

As used herein, “amyloid fibrils” refers to insoluble fibers of amyloidthat are resistant to degradation. Amyloid fibrils can produce symptomsbased on the specific protein or peptide and the tissue and cell type inwhich it has aggregated.

As used herein, “amyloidosis” refers to a disease characterized bysymptoms caused by deposition of amyloid or amyloid fibrils. Amyloidosiscan affect numerous organs including the heart, kidney, liver, spleen,nervous system, and digestive track.

As used herein, “ATTR,” “TTR-related amyloidosis,” “TTR amyloidosis,”“ATTR amyloidosis,” or “amyloidosis associated with TTR” refers toamyloidosis associated with deposition of TTR.

As used herein, “familial amyloid cardiomyopathy” or “FAC” refers to ahereditary transthyretin amyloidosis (ATTR) characterized primarily byrestrictive cardiomyopathy. Congestive heart failure is common in FAC.Average age of onset is approximately 60-70 years of age, with anestimated life expectancy of 4-5 years after diagnosis.

As used herein, “familial amyloid polyneuropathy” or “FAP” refers to ahereditary transthyretin amyloidosis (ATTR) characterized primarily bysensorimotor neuropathy. Autonomic neuropathy is common in FAP. Whileneuropathy is a primary feature, symptoms of FAP may also includecachexia, renal failure, and cardiac disease. Average age of onset ofFAP is approximately 30-50 years of age, with an estimated lifeexpectancy of 5-15 after diagnosis.

As used herein, “wild-type ATTR” and “ATTRwt” refer to ATTR notassociated with a pathological TTR mutation such as T60A, V30M, V30A,V30G, V30L, V122I, V122A, or V122(−). ATTRwt has also been referred toas senile systemic amyloidosis. Onset typically occurs in men aged 60 orhigher with the most common symptoms being congestive heart failure andabnormal heart rhythm such as atrial fibrillation. Additional symptomsinclude consequences of poor heart function such as shortness of breath,fatigue, dizziness, swelling (especially in the legs), nausea, angina,disrupted sleep, and weight loss. A history of carpal tunnel syndromeindicates increased risk for ATTRwt and may in some cases be indicativeof early-stage disease. ATTRwt generally leads to decreasing heartfunction over time but can have a better prognosis than hereditary ATTRbecause wild-type TTR deposits accumulate more slowly. Existingtreatments are similar to other forms of ATTR (other than livertransplantation) and are generally directed to supporting or improvingheart function, ranging from diuretics and limited fluid and salt intaketo anticoagulants, and in severe cases, heart transplants. Nonetheless,like FAC, ATTRwt can result in death from heart failure, sometimeswithin 3-5 years of diagnosis.

Guide sequences useful in the guide RNA compositions and methodsdescribed herein are shown in Table 1 and throughout the application.

As used herein, “hereditary ATTR” refers to ATTR that is associated witha mutation in the sequence of the TTR gene. Known mutations in the TTRgene associated with ATTR include those resulting in TTR withsubstitutions of T60A, V30M, V30A, V30G, V30L, V122I, V122A, or V122(−).

As used herein, “indels” refer to insertion/deletion mutationsconsisting of a number of nucleotides that are either inserted ordeleted at the site of double-stranded breaks (DSBs) in a target nucleicacid.

As used herein, “knockdown” refers to a decrease in expression of aparticular gene product (e.g., protein, mRNA, or both). Knockdown of aprotein can be measured either by detecting protein secreted by tissueor population of cells (e.g., in serum or cell media) or by detectingtotal cellular amount of the protein from a tissue or cell population ofinterest. Methods for measuring knockdown of mRNA are known, and includesequencing of mRNA isolated from a tissue or cell population ofinterest. In some embodiments, “knockdown” may refer to some loss ofexpression of a particular gene product, for example a decrease in theamount of mRNA transcribed or a decrease in the amount of proteinexpressed or secreted by a population of cells (including in vivopopulations such as those found in tissues).

As used herein, “knockout” refers to a loss of expression of aparticular protein in a cell. Knockout can be measured either bydetecting the amount of protein secretion from a tissue or population ofcells (e.g., in serum or cell media) or by detecting total cellularamount of a protein a tissue or a population of cells. In someembodiments, the methods of the disclosure “knockout” TTR in one or morecells (e.g., in a population of cells including in vivo populations suchas those found in tissues). In some embodiments, a knockout is not theformation of mutant TTR protein, for example, created by indels, butrather the complete loss of expression of TTR protein in a cell.

As used herein, “mutant TTR” refers to a gene product of TTR (i.e., theTTR protein) having a change in the amino acid sequence of TTR comparedto the wildtype amino acid sequence of TTR. The human wild-type TTRsequence is available at NCBI Gene ID: 7276; Ensembl: Ensembl:ENSG00000118271. Mutants forms of TTR associated with ATTR, e.g., inhumans, include T60A, V30M, V30A, V30G, V30L, V122I, V122A, or V122(−).

As used herein, “mutant TTR” or “mutant TTR allele” refers to a TTRsequence having a change in the nucleotide sequence of TTR compared tothe wildtype sequence (NCBI Gene ID: 7276; Ensembl: ENSG00000118271).

As used herein, “ribonucleoprotein” (RNP) or “RNP complex” refers to aguide RNA together with an RNA-guided DNA binding agent, such as a Casnuclease, e.g., a Cas cleavase, Cas nickase, or dCas DNA binding agent(e.g., Cas9). In some embodiments, the guide RNA guides the RNA-guidedDNA binding agent such as Cas9 to a target sequence, and the guide RNAhybridizes with and the agent binds to the target sequence; in caseswhere the agent is a cleavase or nickase, binding can be followed bycleaving or nicking.

As used herein, a “target sequence” refers to a sequence of nucleic acidin a target gene that has complementarity to the guide sequence of thegRNA. The interaction of the target sequence and the guide sequencedirects an RNA-guided DNA binding agent to bind, and potentially nick orcleave (depending on the activity of the agent), within the targetsequence.

As used herein, “treatment” refers to any administration or applicationof a therapeutic for disease or disorder in a subject, and includesinhibiting the disease, arresting its development, relieving one or moresymptoms of the disease, curing the disease, or preventing reoccurrenceof one or more symptoms of the disease. For example, treatment of ATTRmay comprise alleviating symptoms of ATTR.

“Modified uridine” is used herein to refer to a nucleoside other thanthymidine with the same hydrogen bond acceptors as uridine and one ormore structural differences from uridine. In some embodiments, amodified uridine is a substituted uridine, i.e., a uridine in which oneor more non-proton substituents (e.g., alkoxy, such as methoxy) takesthe place of a proton. In some embodiments, a modified uridine ispseudouridine. In some embodiments, a modified uridine is a substitutedpseudouridine, i.e., a pseudouridine in which one or more non-protonsubstituents (e.g., alkyl, such as methyl) takes the place of a proton,e.g., N1-methyl pseudouridine. In some embodiments, a modified uridineis any of a substituted uridine, pseudouridine, or a substitutedpseudouridine.

As used herein, a first sequence is considered to “comprise a sequencewith at least X % identity to” a second sequence if an alignment of thefirst sequence to the second sequence shows that X % or more of thepositions of the second sequence in its entirety are matched by thefirst sequence. For example, the sequence AAGA comprises a sequence with100% identity to the sequence AAG because an alignment would give 100%identity in that there are matches to all three positions of the secondsequence. The differences between RNA and DNA (generally the exchange ofuridine for thymidine or vice versa) and the presence of nucleosideanalogs such as modified uridines do not contribute to differences inidentity or complementarity among polynucleotides as long as therelevant nucleotides (such as thymidine, uridine, or modified uridine)have the same complement (e.g., adenosine for all of thymidine, uridine,or modified uridine; another example is cytosine and 5-methylcytosine,both of which have guanosine as a complement). Thus, for example, thesequence 5′-AXG where X is any modified uridine, such as pseudouridine,N1-methyl pseudouridine, or 5-methoxyuridine, is considered 100%identical to AUG in that both are perfectly complementary to the samesequence (5′-CAU). Exemplary alignment algorithms are the Smith-Watermanand Needleman-Wunsch algorithms, which are well-known in the art. Oneskilled in the art will understand what choice of algorithm andparameter settings are appropriate for a given pair of sequences to bealigned; for sequences of generally similar length and expectedidentity >50% for amino acids or >75% for nucleotides, theNeedleman-Wunsch algorithm with default settings of the Needleman-Wunschalgorithm interface provided by the EBI at the www.ebi.ac.uk web serverare generally appropriate.

The term “about” or “approximately” means an acceptable error for aparticular value as determined by one of ordinary skill in the art,which depends in part on how the value is measured or determined.

II. Compositions

A. Compositions Comprising Guide RNA (gRNAs)

Provided herein are compositions useful for editing the TTR gene, e.g.,using a guide RNA with an RNA-guided DNA binding agent (e.g., aCRISPR/Cas system). The compositions may be administered to subjectshaving wild-type or non-wild type TTR gene sequences, such as, forexample, subjects with ATTR, which may be ATTR wt or a hereditary orfamilial form of ATTR. Guide sequences targeting the TTR gene are shownin Table 1 at SEQ ID Nos: 5-82.

TABLE 1TTR targeted guide sequences, nomenclature, chromosomal coordinates, andsequence. SEQ ID Guide De- Chromosomal No. ID scription Species LocationGuide Sequences*  5 CR003335 TTR Human chr18:315919 CUGCUCCUCCUCUGCCUUGC(Exon 1) 17-31591937  6 CR003336 TTR Human chr18:315919CCUCCUCUGCCUUGCUGGAC (Exon 1) 22-31591942  7 CR003337 TTR Humanchr18:315919 CCAGUCCAGCAAGGCAGAGG (Exon 1) 25-31591945  8 CR003338 TTRHuman chr18:315919 AUACCAGUCCAGCAAGGCAG (Exon 1) 28-31591948  9 CR003339TTR Human chr18:315919 ACACAAAUACCAGUCCAGCA (Exon 1) 34-31591954 10CR003340 TTR Human chr18:315919 UGGACUGGUAUUUGUGUCUG (Exon 1)37-31591957 11 CR003341 TTR Human chr18:315919 CUGGUAUUUGUGUCUGAGGC(Exon 1) 41-31591961 12 CR003342 TTR Human chr18:315928CUUCUCUACACCCAGGGCAC (Exon 2) 80-31592900 13 CR003343 TTR Humanchr18:315929 CAGAGGACACUUGGAUUCAC (Exon 2) 02-31592922 14 CR003344 TTRHuman chr18:315929 UUUGACCAUCAGAGGACACU (Exon 2) 11-31592931 15 CR003345TTR Human chr18:315929 UCUAGAACUUUGACCAUCAG (Exon 2) 19-31592939 16CR003346 TTR Human chr18:315929 AAAGUUCUAGAUGCUGUCCG (Exon 2)28-31592948 17 CR003347 TTR Human chr18:315929 CAUUGAUGGCAGGACUGCCU(Exon 2) 48-31592968 18 CR003348 TTR Human chr18:315929AGGCAGUCCUGCCAUCAAUG (Exon 2) 48-31592968 19 CR003349 TTR Humanchr18:315929 UGCACGGCCACAUUGAUGGC (Exon 2) 58-31592978 20 CR003350 TTRHuman chr18:315929 CACAUGCACGGCCACAUUGA (Exon 2) 62-31592982 21 CR003351TTR Human chr18:315929 AGCCUUUCUGAACACAUGCA (Exon 2) 74-31592994 22CR003352 TTR Human chr18:315929 GAAAGGCUGCUGAUGACACC (Exon 2)86-31593006 23 CR003353 TTR Human chr18:315929 AAAGGCUGCUGAUGACACCU(Exon 2) 87-31593007 24 CR003354 TTR Human chr18:315930ACCUGGGAGCCAUUUGCCUC (Exon 2) 03-31593023 25 CR003355 TTR Humanchr18:315930 CCCAGAGGCAAAUGGCUCCC (Exon 2) 07-31593027 26 CR003356 TTRHuman chr18:315930 GCAACUUACCCAGAGGCAAA (Exon 2) 15-31593035 27 CR003357TTR Human chr18:315930 UUCUUUGGCAACUUACCCAG (Exon 2) 22-31593042 28CR003358 TTR Human chr18:315951 AUGCAGCUCUCCAGACUCAC (Exon 3)27-31595147 29 CR003359 TTR Human chr18:315951 AGUGAGUCUGGAGAGCUGCA(Exon 3) 26-31595146 30 CR003360 TTR Human chr18:315951GUGAGUCUGGAGAGCUGCAU (Exon 3) 27-31595147 31 CR003361 TTR Humanchr18:315951 GCUGCAUGGGCUCACAACUG (Exon 3) 40-31595160 32 CR003362 TTRHuman chr18:315951 GCAUGGGCUCACAACUGAGG (Exon 3) 43-31595163 33 CR003363TTR Human chr18:315951 ACUGAGGAGGAAUUUGUAGA (Exon 3) 56-31595176 34CR003364 TTR Human chr18:315951 CUGAGGAGGAAUUUGUAGAA (Exon 3)57-31595177 35 CR003365 TTR Human chr18:315951 UGUAGAAGGGAUAUACAAAG(Exon 3) 70-31595190 36 CR003366 TTR Human chr18:315951AAAUAGACACCAAAUCUUAC (Exon 3) 93-31595213 37 CR003367 TTR Humanchr18:315951 AGACACCAAAUCUUACUGGA (Exon 3) 97-31595217 38 CR003368 TTRHuman chr18:315952 AAGUGCCUUCCAGUAAGAUU (Exon 3) 05-31595225 39 CR003369TTR Human chr18:315952 CUCUGCAUGCUCAUGGAAUG (Exon 3) 35-31595255 40CR003370 TTR Human chr18:315952 CCUCUGCAUGCUCAUGGAAU (Exon 3)36-31595256 41 CR003371 TTR Human chr18:315952 ACCUCUGCAUGCUCAUGGAA(Exon 3) 37-31595257 42 CR003372 TTR Human chr18:315952UACUCACCUCUGCAUGCUCA (Exon 3) 42-31595262 43 CR003373 TTR Humanchr18:315985 GUAUUCACAGCCAACGACUC (Exon 4) 70-31598590 44 CR003374 TTRHuman chr18:315985 GCGGCGGGGGCCGGAGUCGU (Exon 4) 83-31598603 45 CR003375TTR Human chr18:315985 AAUGGUGUAGCGGCGGGGGC (Exon 4) 92-31598612 46CR003376 TTR Human chr18:315985 CGGCAAUGGUGUAGCGGCGG (Exon 4)96-31598616 47 CR003377 TTR Human chr18:315985 GCGGCAAUGGUGUAGCGGCG(Exon 4) 97-31598617 48 CR003378 TTR Human chr18:315985GGCGGCAAUGGUGUAGCGGC (Exon 4) 98-31598618 49 CR003379 TTR Humanchr18:315985 GGGCGGCAAUGGUGUAGCGG (Exon 4) 99-31598619 50 CR003380 TTRHuman chr18:315986 GCAGGGCGGCAAUGGUGUAG (Exon 4) 02-31598622 51 CR003381TTR Human chr18:315986 GGGGCUCAGCAGGGCGGCAA (Exon 4) 10-31598630 52CR003382 TTR Human chr18:315986 GGAGUAGGGGCUCAGCAGGG (Exon 4)16-31598636 53 CR003383 TTR Human chr18:315986 AUAGGAGUAGGGGCUCAGCA(Exon 4) 19-31598639 54 CR003384 TTR Human chr18:315986AAUAGGAGUAGGGGCUCAGC (Exon 4) 20-31598640 55 CR003385 TTR Humanchr18:315986 CCCCUACUCCUAUUCCACCA (Exon 4) 26-31598646 56 CR003386 TTRHuman chr18:315986 CCGUGGUGGAAUAGGAGUAG (Exon 4) 29-31598649 57 CR003387TTR Human chr18:315986 GCCGUGGUGGAAUAGGAGUA (Exon 4) 30-31598650 58CR003388 TTR Human chr18:315986 GACGACAGCCGUGGUGGAAU (Exon 4)37-31598657 59 CR003389 TTR Human chr18:315986 AUUGGUGACGACAGCCGUGG(Exon 4) 43-31598663 60 CR003390 TTR Human chr18:315986GGGAUUGGUGACGACAGCCG (Exon 4) 46-31598666 61 CR003391 TTR Humanchr18:315986 GGCUGUCGUCACCAAUCCCA (Exon 4) 47-31598667 62 CR003392 TTRHuman chr18:315986 AGUCCCUCAUUCCUUGGGAU (Exon 4) 61-31598681 63 CR005298TTR Human chr18:315918 UCCACUCAUUCUUGGCAGGA (Exon 1) 83-31591903 64CR005299 TTR Human chr18:315986 AGCCGUGGUGGAAUAGGAGU (Exon 4)31-31598651 65 CR005300 TTR Human chr18:315919 UCACAGAAACACUCACCGUA(Exon 1) 67-31591987 66 CR005301 TTR Human chr18:315919GUCACAGAAACACUCACCGU (Exon 1) 68-31591988 67 CR005302 TTR Humanchr18:315928 ACGUGUCUUCUCUACACCCA (Exon 2) 74-31592894 68 CR005303 TTRHuman chr18:315929 UGAAUCCAAGUGUCCUCUGA (Exon 2) 03-31592923 69 CR005304TTR Human chr18:315929 GGCCGUGCAUGUGUUCAGAA (Exon 2) 69-31592989 70CR005305 TTR Human chr18:315951 UAUAGGAAAACCAGUGAGUC (Exon 3)14-31595134 71 CR005306 TTR Human chr18:315952 AAAUCUUACUGGAAGGCACU(Exon 3) 04-31595224 72 CR005307 TTR Human chr18:315985UGUCUGUCUUCUCUCAUAGG (Exon 4) 48-31598568 73 CR000689 TTR Cynochr18:506815 ACACAAAUACCAGUCCAGCG 33-50681553 74 CR005364 TTR Cynochr18:506804 AAAGGCUGCUGAUGAGACCU 81-50680501 75 CR005365 TTR Cynochr18:506805 CAUUGACAGCAGGACUGCCU 20-50680540 76 CR005366 TTR Cynochr18:506815 AUACCAGUCCAGCGAGGCAG 39-50681559 77 CR005367 TTR Cynochr18:506815 CCAGUCCAGCGAGGCAGAGG 42-50681562 78 CR005368 TTR Cynochr18:506815 CCUCCUCUGCCUCGCUGGAC 45-50681565 79 CR005369 TTR Cynochr18:506805 AAAGUUCUAGAUGCCGUCCG 40-50680560 80 CR005370 TTR Cynochr18:506805 ACUUGUCUUCUCUAUACCCA 94-50680614 81 CR005371 TTR Cynochr18:506782 AAGUGACUUCCAGUAAGAUU 16-50678236 82 CR005372 TTR Cynochr18:506804 AAAAGGCUGCUGAUGAGACC 82-50680502

Each of the Guide Sequences above may further comprise additionalnucleotides to form a crRNA, e.g., with the following exemplarynucleotide sequence following the Guide Sequence at its 3′ end:GUUUUAGAGCUAUGCUGUUUUG (SEQ ID NO: 126). In the case of a sgRNA, theabove Guide Sequences may further comprise additional nucleotides toform a sgRNA, e.g., with the following exemplary nucleotide sequencefollowing the 3′ end of the Guide Sequence:GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 125) in 5′ to 3′ orientation.

In some embodiments, the sgRNA is modified. In some embodiments, thesgRNA comprises the modification pattern shown below in SEQ ID NO: 3,where N is any natural or non-natural nucleotide, and where the totalityof the N's comprise a guide sequence as described herein and themodified sgRNA comprises the following sequence:mN*mN*mN*GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU (SEQ ID NO: 3), where“N” may be any natural or non-natural nucleotide. For example,encompassed herein is SEQ ID NO: 3, where the N's are replaced with anyof the guide sequences disclosed herein. The modifications remain asshown in SEQ ID NO: 3 despite the substitution of N's for thenucleotides of a guide. That is, although the nucleotides of the guidereplace the “N's”, the first three nucleotides are 2′OMe modified andthere are phosphorothioate linkages between the first and secondnucleotides, the second and third nucleotides and the third and fourthnucleotides.

In some embodiments, any one of the sequences recited in Table 2 isencompassed.

TABLE 2 TTR targeted sgRNA sequences SEQ ID Guide Target and No. IDDescription Species Sequence  87 G000480 TTR HumanmA*mA*mA*GGCUGCUGAUGACACCUGU sgRNA UUUAGAmGmCmUmAmGmAmAmAmUmA modifiedmGmCAAGUUAAAAUAAGGCUAGUCCGU sequence UAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU  88 G000481 TTR HumanmU*mC*mU*AGAACUUUGACCAUCAGGU sgRNA UUUAGAmGmCmUmAmGmAmAmAmUmA modifiedmGmCAAGUUAAAAUAAGGCUAGUCCGU sequence UAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU  89 G000482 TTR HumanmU*mG*mU*AGAAGGGAUAUACAAAGG sgRNA UUUUAGAmGmCmUmAmGmAmAmAmUm modifiedAmGmCAAGUUAAAAUAAGGCUAGUCCG sequence UUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmC mGmGmUmGmCmU*mU*mU*mU  90 G000483 TTR HumanmU*mC*mC*ACUCAUUCUUGGCAGGAGU sgRNA UUUAGAmGmCmUmAmGmAmAmAmUmA modifiedmGmCAAGUUAAAAUAAGGCUAGUCCGU sequence UAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU  91 G000484 TTR HumanmA*mG*mA*CACCAAAUCUUACUGGAGU sgRNA UUUAGAmGmCmUmAmGmAmAmAmUmA modifiedmGmCAAGUUAAAAUAAGGCUAGUCCGU sequence UAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU  92 G000485 TTR HumanmC*mC*mU*CCUCUGCCUUGCUGGACGU sgRNA UUUAGAmGmCmUmAmGmAmAmAmUmA modifiedmGmCAAGUUAAAAUAAGGCUAGUCCGU sequence UAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU  93 G000486 TTR HumanmA*mC*mA*CAAAUACCAGUCCAGCAGU sgRNA UUUAGAmGmCmUmAmGmAmAmAmUmA modifiedmGmCAAGUUAAAAUAAGGCUAGUCCGU sequence UAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU  94 G000487 TTR HumanmU*mU*mC*UUUGGCAACUUACCCAGGU sgRNA UUUAGAmGmCmUmAmGmAmAmAmUmA modifiedmGmCAAGUUAAAAUAAGGCUAGUCCGU sequence UAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU  95 G000488 TTR HumanmA*mA*mA*GUUCUAGAUGCUGUCCGGU sgRNA UUUAGAmGmCmUmAmGmAmAmAmUmA modifiedmGmCAAGUUAAAAUAAGGCUAGUCCGU sequence UAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU  96 G000489 TTR HumanmU*mU*mU*GACCAUCAGAGGACACUGU sgRNA UUUAGAmGmCmUmAmGmAmAmAmUmA modifiedmGmCAAGUUAAAAUAAGGCUAGUCCGU sequence UAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU  97 G000490 TTR HumanmA*mA*mA*UAGACACCAAAUCUUACGU sgRNA UUUAGAmGmCmUmAmGmAmAmAmUmA modifiedmGmCAAGUUAAAAUAAGGCUAGUCCGU sequence UAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU  98 G000491 TTR HumanmA*mU*mA*CCAGUCCAGCAAGGCAGGU sgRNA UUUAGAmGmCmUmAmGmAmAmAmUmA modifiedmGmCAAGUUAAAAUAAGGCUAGUCCGU sequence UAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU  99 G000492 TTR HumanmC*mU*mU*CUCUACACCCAGGGCACGU sgRNA UUUAGAmGmCmUmAmGmAmAmAmUmA modifiedmGmCAAGUUAAAAUAAGGCUAGUCCGU sequence UAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU 100 G000493 TTR HumanmA*mA*mG*UGCCUUCCAGUAAGAUUGU sgRNA UUUAGAmGmCmUmAmGmAmAmAmUmA modifiedmGmCAAGUUAAAAUAAGGCUAGUCCGU sequence UAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU 101 G000494 TTR HumanmG*mU*mG*AGUCUGGAGAGCUGCAUGU sgRNA UUUAGAmGmCmUmAmGmAmAmAmUmA modifiedmGmCAAGUUAAAAUAAGGCUAGUCCGU sequence UAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU 102 G000495 TTR HumanmC*mA*mG*AGGACACUUGGAUUCACGU sgRNA UUUAGAmGmCmUmAmGmAmAmAmUmA modifiedmGmCAAGUUAAAAUAAGGCUAGUCCGU sequence UAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU 103 G000496 TTR HumanmG*mG*mC*CGUGCAUGUGUUCAGAAGU sgRNA UUUAGAmGmCmUmAmGmAmAmAmUmA modifiedmGmCAAGUUAAAAUAAGGCUAGUCCGU sequence UAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU 104 G000497 TTR HumanmC*mU*mG*CUCCUCCUCUGCCUUGCGU sgRNA UUUAGAmGmCmUmAmGmAmAmAmUmA modifiedmGmCAAGUUAAAAUAAGGCUAGUCCGU sequence UAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU 105 G000498 TTR HumanmA*mG*mU*GAGUCUGGAGAGCUGCAGU sgRNA UUUAGAmGmCmUmAmGmAmAmAmUmA modifiedmGmCAAGUUAAAAUAAGGCUAGUCCGU sequence UAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU 106 G000499 TTR HumanmU*mG*mA*AUCCAAGUGUCCUCUGAGU sgRNA UUUAGAmGmCmUmAmGmAmAmAmUmA modifiedmGmCAAGUUAAAAUAAGGCUAGUCCGU sequence UAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU 107 G000500 TTR HumanmC*mC*mA*GUCCAGCAAGGCAGAGGGU sgRNA UUUAGAmGmCmUmAmGmAmAmAmUmA modifiedmGmCAAGUUAAAAUAAGGCUAGUCCGU sequence UAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU 108 G000501 TTR HumanmU*mC*mA*CAGAAACACUCACCGUAGU sgRNA UUUAGAmGmCmUmAmGmAmAmAmUmA modifiedmGmCAAGUUAAAAUAAGGCUAGUCCGU sequence UAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU 109 G000567 TTR HumanmG*mA*mA*AGGCUGCUGAUGACACCGU sgRNA UUUAGAmGmCmUmAmGmAmAmAmUmA modifiedmGmCAAGUUAAAAUAAGGCUAGUCCGU sequence UAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU 110 G000568 TTR HumanmG*mG*mC*UGUCGUCACCAAUCCCAGU sgRNA UUUAGAmGmCmUmAmGmAmAmAmUmA modifiedmGmCAAGUUAAAAUAAGGCUAGUCCGU sequence UAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU 111 G000570 TTR HumanmC*mA*mU*UGAUGGCAGGACUGCCUGU sgRNA UUUAGAmGmCmUmAmGmAmAmAmUmA modifiedmGmCAAGUUAAAAUAAGGCUAGUCCGU sequence UAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU 112 G000571 TTR HumanmG*mU*mC*ACAGAAACACUCACCGUGU sgRNA UUUAGAmGmCmUmAmGmAmAmAmUmA modifiedmGmCAAGUUAAAAUAAGGCUAGUCCGU sequence UAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU 113 G000572 TTR HumanmC*mC*mC*CUACUCCUAUUCCACCAGU sgRNA UUUAGAmGmCmUmAmGmAmAmAmUmA modifiedmGmCAAGUUAAAAUAAGGCUAGUCCGU sequence UAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU 114 G000502 TTR CynoCyno mA*mC*mA*CAAAUACCAGUCCAGCGGU specific UUUAGAmGmCmUmAmGmAmAmAmUmAsgRNA mGmCAAGUUAAAAUAAGGCUAGUCCGU modified UAUCAmAmCmUmUmGmAmAmAmAmAmsequence GmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU 115 G000503TTR Cyno Cyno mA*mA*mA*AGGCUGCUGAUGAGACCGU specificUUUAGAmGmCmUmAmGmAmAmAmUmA sgRNA mGmCAAGUUAAAAUAAGGCUAGUCCGU modifiedUAUCAmAmCmUmUmGmAmAmAmAmAm sequence GmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU 116 G000504 TTR Cyno CynomA*mA*mA*GGCUGCUGAUGAGACCUGU specific UUUAGAmGmCmUmAmGmAmAmAmUmA sgRNAmGmCAAGUUAAAAUAAGGCUAGUCCGU modified UAUCAmAmCmUmUmGmAmAmAmAmAm sequenceGmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU 117 G000505 TTR CynoCyno mC*mA*mU*UGACAGCAGGACUGCCUGU specific UUUAGAmGmCmUmAmGmAmAmAmUmAsgRNA mGmCAAGUUAAAAUAAGGCUAGUCCGU modified UAUCAmAmCmUmUmGmAmAmAmAmAmsequence GmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU 118 G000506TTR Cyno Cyno mA*mU*mA*CCAGUCCAGCGAGGCAGGU specificUUUAGAmGmCmUmAmGmAmAmAmUmA sgRNA mGmCAAGUUAAAAUAAGGCUAGUCCGU modifiedUAUCAmAmCmUmUmGmAmAmAmAmAm sequence GmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU 119 G000507 TTR Cyno CynomC*mC*mA*GUCCAGCGAGGCAGAGGGU specific UUUAGAmGmCmUmAmGmAmAmAmUmA sgRNAmGmCAAGUUAAAAUAAGGCUAGUCCGU modified UAUCAmAmCmUmUmGmAmAmAmAmAm sequenceGmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU 120 G000508 TTR CynoCyno mC*mC*mU*CCUCUGCCUCGCUGGACGU specific UUUAGAmGmCmUmAmGmAmAmAmUmAsgRNA mGmCAAGUUAAAAUAAGGCUAGUCCGU modified UAUCAmAmCmUmUmGmAmAmAmAmAmsequence GmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU 121 G000509TTR Cyno Cyno mA*mA*mA*GUUCUAGAUGCCGUCCGGU specificUUUAGAmGmCmUmAmGmAmAmAmUmA sgRNA mGmCAAGUUAAAAUAAGGCUAGUCCGU modifiedUAUCAmAmCmUmUmGmAmAmAmAmAm sequence GmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU 122 G000510 TTR Cyno CynomA*mC*mU*UGUCUUCUCUAUACCCAGU specific UUUAGAmGmCmUmAmGmAmAmAmUmA sgRNAmGmCAAGUUAAAAUAAGGCUAGUCCGU modified UAUCAmAmCmUmUmGmAmAmAmAmAm sequenceGmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU 123 G000511 TTR CynoCyno mA*mA*mG*UGACUUCCAGUAAGAUUGU specific UUUAGAmGmCmUmAmGmAmAmAmUmAsgRNA mGmCAAGUUAAAAUAAGGCUAGUCCGU modified UAUCAmAmCmUmUmGmAmAmAmAmAmsequence GmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU 124 G000282 TTRMouse mU*mU*mA*CAGCCACGUCUACAGCAGU UUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGU UAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU * = PS linkage;′m′ = 2′-O-Me nucleotide

An alignment mapping of the Guide IDs with the corresponding sgRNA IDsas well as homology to the cyno genome and cyno matched guide IDs areprovided in Table 3.

TABLE 3 TTR targeted guide sequence ID mapping and Cyno Homology De-Human Human Number Cyno Cyno scrip- Dual Single Mismatches to MatchedMatched tion Guide ID Guide ID Cyno Genome dgRNA ID sgRNA ID TTRCR003335 G000497 1 TTR CR003336 G000485 1 CR005368 G000508 TTR CR003337G000500 1 CR005367 G000507 TTR CR003338 G000491 1 CR005366 G000506 TTRCR003339 G000486 1 CR000689 G000502 TTR CR003340 0 TTR CR003341 0 TTRCR003342 G000492 no PAM in cyno TTR CR003343 G000495 no PAM in cyno TTRCR003344 G000489 0 TTR CR003345 G000481 0 TTR CR003346 G000488 1CR005369 G000509 TTR CR003347 G000570 2 CR005365 G000505 TTR CR003348 2TTR CR003349 >3  TTR CR003350 no PAM in cyno TTR CR003351 no PAM in cynoTTR CR003352 G000567 2 CR005372 G000503 TTR CR003353 G000480 1 CR005364G000504 TTR CR003354 1 TTR CR003355 1 TTR CR003356 3 TTR CR003357G000487 >3  TTR CR003358 0 TTR CR003359 G000498 0 TTR CR003360 G000494 0TTR CR003361 0 TTR CR003362 0 TTR CR003363 0 TTR CR003364 0 TTR CR003365G000482 0 TTR CR003366 G000490 0 TTR CR003367 G000484 no PAM in cyno TTRCR003368 G000493 1 CR005371 G000511 TTR CR003369 0 TTR CR003370 0 TTRCR003371 0 TTR CR003372 0 TTR CR003373 1 TTR CR003374 2 TTR CR003375 2TTR CR003376 2 TTR CR003377 2 TTR CR003378 2 TTR CR003379 2 TTR CR0033801 TTR CR003381 1 TTR CR003382 0 TTR CR003383 0 TTR CR003384 0 TTRCR003385 G000572 0 TTR CR003386 0 TTR CR003387 0 TTR CR003388 0 TTRCR003389 G000569 0 TTR CR003390 0 TTR CR003391 G000568 0 TTR CR003392 0TTR CR005298 G000483 1 TTR CR005299 0 TTR CR005300 G000501 no PAM incyno TTR CR005301 G000571 0 TTR CR005302 2 CR005370 G000510 TTR CR005303G000499 0 TTR CR005304 G000496 >3  TTR CR005305 0 TTR CR005306 1 TTRCR005307 0

In some embodiments, the invention provides a composition comprising oneor more guide RNA (gRNA) comprising guide sequences that direct anRNA-guided DNA binding agent, which can be a nuclease (e.g., a Casnuclease such as Cas9), to a target DNA sequence in TTR. The gRNA maycomprise a crRNA comprising a guide sequence shown in Table 1. The gRNAmay comprise a crRNA comprising 17, 18, 19, or 20 contiguous nucleotidesof a guide sequence shown in Table 1. In some embodiments, the gRNAcomprises a crRNA comprising a sequence with about 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99%, or 100% identity to at least 17, 18, 19, or 20contiguous nucleotides of a guide sequence shown in Table 1. In someembodiments, the gRNA comprises a crRNA comprising a sequence with about75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a guidesequence shown in Table 1. The gRNA may further comprise a trRNA. Ineach composition and method embodiment described herein, the crRNA andtrRNA may be associated as a single RNA (sgRNA), or may be on separateRNAs (dgRNA). In the context of sgRNAs, the crRNA and trRNA componentsmay be covalently linked, e.g., via a phosphodiester bond or othercovalent bond.

In each of the composition, use, and method embodiments describedherein, the guide RNA may comprise two RNA molecules as a “dual guideRNA” or “dgRNA”. The dgRNA comprises a first RNA molecule comprising acrRNA comprising, e.g., a guide sequence shown in Table 1, and a secondRNA molecule comprising a trRNA. The first and second RNA molecules maynot be covalently linked, but may form a RNA duplex via the base pairingbetween portions of the crRNA and the trRNA.

In each of the composition, use, and method embodiments describedherein, the guide RNA may comprise a single RNA molecule as a “singleguide RNA” or “sgRNA”. The sgRNA may comprise a crRNA (or a portionthereof) comprising a guide sequence shown in Table 1 covalently linkedto a trRNA. The sgRNA may comprise 17, 18, 19, or 20 contiguousnucleotides of a guide sequence shown in Table 1. In some embodiments,the crRNA and the trRNA are covalently linked via a linker. In someembodiments, the sgRNA forms a stem-loop structure via the base pairingbetween portions of the crRNA and the trRNA. In some embodiments, thecrRNA and the trRNA are covalently linked via one or more bonds that arenot a phosphodiester bond.

In some embodiments, the trRNA may comprise all or a portion of a trRNAsequence derived from a naturally-occurring CRISPR/Cas system. In someembodiments, the trRNA comprises a truncated or modified wild typetrRNA. The length of the trRNA depends on the CRISPR/Cas system used. Insome embodiments, the trRNA comprises or consists of 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90,100, or more than 100 nucleotides. In some embodiments, the trRNA maycomprise certain secondary structures, such as, for example, one or morehairpin or stem-loop structures, or one or more bulge structures.

In some embodiments, the invention provides a composition comprising oneor more guide RNAs comprising a guide sequence of any one of SEQ ID NOs:5-82.

In one aspect, the invention provides a composition comprising a gRNAthat comprises a guide sequence that is at least 99%, 98%, 97%, 96%,95%, 94%, 93%, 92%, 91%, or 90% identical to any of the nucleic acids ofSEQ ID NOs: 5-82.

In other embodiments, the composition comprises at least one, e.g., atleast two gRNA's comprising guide sequences selected from any two ormore of the guide sequences of SEQ ID NOs: 5-82. In some embodiments,the composition comprises at least two gRNA's that each comprise a guidesequence at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90%identical to any of the nucleic acids of SEQ ID NOs: 5-82.

In some embodiments, the gRNA is a sgRNA comprising any one of thesequences shown in Table 2 (SEQ ID Nos. 87-124). In some embodiments,the gRNA is a sgRNA comprising any one of the sequences shown in Table 2(SEQ ID Nos. 87-124, but without the modifications as shown (i.e.,unmodified SEQ ID Nos. 87-124). In some embodiments, the sgRNA comprisesa sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%,or 90% identical to any of the nucleic acids of SEQ ID Nos. 87-124. Insome embodiments, the sgRNA comprises a sequence that is at least 99%,98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to any of thenucleic acids of SEQ ID Nos. 87-124, but without the modifications asshown (i.e., unmodified SEQ ID Nos. 87-124). In some embodiments, thesgRNA comprises any one of the guide sequences shown in Table 1 in placeof the guide sequences shown in the sgRNA sequences of Table 2 at SEQ IDNos: 87-124, with or without the modifications.

The guide RNA compositions of the present invention are designed torecognize (e.g., hybridize to) a target sequence in the TTR gene. Forexample, the TTR target sequence may be recognized and cleaved by aprovided Cas cleavase comprising a guide RNA. In some embodiments, anRNA-guided DNA binding agent, such as a Cas cleavase, may be directed bya guide RNA to a target sequence of the TTR gene, where the guidesequence of the guide RNA hybridizes with the target sequence and theRNA-guided DNA binding agent, such as a Cas cleavase, cleaves the targetsequence.

In some embodiments, the selection of the one or more guide RNAs isdetermined based on target sequences within the TTR gene.

Without being bound by any particular theory, mutations (e.g.,frameshift mutations resulting from indels occurring as a result of anuclease-mediated DSB) in certain regions of the gene may be lesstolerable than mutations in other regions of the gene, thus the locationof a DSB is an important factor in the amount or type of proteinknockdown that may result. In some embodiments, a gRNA complementary orhaving complementarity to a target sequence within TTR is used to directthe RNA-guided DNA binding agent to a particular location in the TTRgene. In some embodiments, gRNAs are designed to have guide sequencesthat are complementary or have complementarity to target sequences inexon 1, exon 2, exon 3, or exon 4 of TTR.

In some embodiments, the guide sequence is at least 99%, 98%, 97%, 96%,95%, 94%, 93%, 92%, 91%, or 90% identical to a target sequence presentin the human TTR gene. In some embodiments, the target sequence may becomplementary to the guide sequence of the guide RNA. In someembodiments, the degree of complementarity or identity between a guidesequence of a guide RNA and its corresponding target sequence may be atleast 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In someembodiments, the target sequence and the guide sequence of the gRNA maybe 100% complementary or identical. In other embodiments, the targetsequence and the guide sequence of the gRNA may contain at least onemismatch. For example, the target sequence and the guide sequence of thegRNA may contain 1, 2, 3, or 4 mismatches, where the total length of theguide sequence is 20. In some embodiments, the target sequence and theguide sequence of the gRNA may contain 1-4 mismatches where the guidesequence is 20 nucleotides.

In some embodiments, a composition or formulation disclosed hereincomprises an mRNA comprising an open reading frame (ORF) encoding anRNA-guided DNA binding agent, such as a Cas nuclease as describedherein. In some embodiments, an mRNA comprising an ORF encoding anRNA-guided DNA binding agent, such as a Cas nuclease, is provided, used,or administered.

In some embodiments, the RNA-guided DNA-binding agent is a Class 2 Casnuclease. In some embodiments, the RNA-guided DNA-binding agent hascleavase activity, which can also be referred to as double-strandendonuclease activity. In some embodiments, the RNA-guided DNA-bindingagent comprises a Cas nuclease, such as a Class 2 Cas nuclease (whichmay be, e.g., a Cas nuclease of Type II, V, or VI). Class 2 Casnucleases include, for example, Cas9, Cpf1, C2c1, C2c2, and C2c3proteins and modifications thereof. Examples of Cas9 nucleases includethose of the type II CRISPR systems of S. pyogenes, S. aureus, and otherprokaryotes (see, e.g., the list in the next paragraph), and modified(e.g., engineered or mutant) versions thereof. See, e.g., US2016/0312198A1; US 2016/0312199 A1. Other examples of Cas nucleases include a Csm orCmr complex of a type III CRISPR system or the Cas10, Csm1, or Cmr2subunit thereof; and a Cascade complex of a type I CRISPR system, or theCas3 subunit thereof. In some embodiments, the Cas nuclease may be froma Type-IIA, Type-IIB, or Type-IIC system. For discussion of variousCRISPR systems and Cas nucleases see, e.g., Makarova et al., NAT. REV.MICROBIOL. 9:467-477 (2011); Makarova et al., NAT. REV. MICROBIOL, 13:722-36 (2015); Shmakov et al., MOLECULAR CELL, 60:385-397 (2015).

Non-limiting exemplary species that the Cas nuclease can be derived frominclude Streptococcus pyogenes, Streptococcus thermophilus,Streptococcus sp., Staphylococcus aureus, Listeria innocua,Lactobacillus gasseri, Francisella novicida, Wolinella succinogenes,Sutterella wadsworthensis, Gammaproteobacterium, Neisseria meningitidis,Campylobacter jejuni, Pasteurella multocida, Fibrobacter succinogene,Rhodospirillum rubrum, Nocardiopsis dassonvillei, Streptomycespristinaespiralis, Streptomyces viridochromogenes, Streptomycesviridochromogenes, Streptosporangium roseum, Streptosporangium roseum,Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillusselenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii,Lactobacillus salivarius, Lactobacillus buchneri, Treponema denticola,Microscilla marina, Burkholderiales bacterium, Polaromonasnaphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothecesp., Microcystis aeruginosa, Synechococcus sp., Acetohalobiumarabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, CandidatusDesulforudis, Clostridium botulinum, Clostridium difficile, Finegoldiamagna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum,Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatiumvinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcuswatsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer,Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena,Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp.,Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotogamobilis, Thermosipho africanus, Streptococcus pasteurianus, Neisseriacinerea, Campylobacter lari, Parvibaculum lavamentivorans,Corynebacterium diphtheria, Acidaminococcus sp., Lachnospiraceaebacterium ND2006, and Acaryochloris marina.

In some embodiments, the Cas nuclease is the Cas9 nuclease fromStreptococcus pyogenes. In some embodiments, the Cas nuclease is theCas9 nuclease from Streptococcus thermophilus. In some embodiments, theCas nuclease is the Cas9 nuclease from Neisseria meningitidis. In someembodiments, the Cas nuclease is the Cas9 nuclease is fromStaphylococcus aureus. In some embodiments, the Cas nuclease is the Cpf1nuclease from Francisella novicida. In some embodiments, the Casnuclease is the Cpf1 nuclease from Acidaminococcus sp. In someembodiments, the Cas nuclease is the Cpf1 nuclease from Lachnospiraceaebacterium ND2006. In further embodiments, the Cas nuclease is the Cpf1nuclease from Francisella tularensis, Lachnospiraceae bacterium,Butyrivibrio proteoclasticus, Peregrinibacteria bacterium, Parcubacteriabacterium, Smithella, Acidaminococcus, Candidatus Methanoplasmatermitum, Eubacterium eligens, Moraxella bovoculi, Leptospira inadai,Porphyromonas crevioricanis, Prevotella disiens, or Porphyromonasmacacae. In certain embodiments, the Cas nuclease is a Cpf1 nucleasefrom an Acidaminococcus or Lachnospiraceae.

Wild type Cas9 has two nuclease domains: RuvC and HNH. The RuvC domaincleaves the non-target DNA strand, and the HNH domain cleaves the targetstrand of DNA. In some embodiments, the Cas9 nuclease comprises morethan one RuvC domain and/or more than one HNH domain. In someembodiments, the Cas9 nuclease is a wild type Cas9. In some embodiments,the Cas9 is capable of inducing a double strand break in target DNA. Incertain embodiments, the Cas nuclease may cleave dsDNA, it may cleaveone strand of dsDNA, or it may not have DNA cleavase or nickaseactivity. An exemplary Cas9 amino acid sequence is provided as SEQ IDNO: 203. An exemplary Cas9 mRNA ORF sequence, which includes start andstop codons, is provided as SEQ ID NO: 204. An exemplary Cas9 mRNAcoding sequence, suitable for inclusion in a fusion protein, is providedas SEQ ID NO: 210.

In some embodiments, chimeric Cas nucleases are used, where one domainor region of the protein is replaced by a portion of a differentprotein. In some embodiments, a Cas nuclease domain may be replaced witha domain from a different nuclease such as FokI. In some embodiments, aCas nuclease may be a modified nuclease.

In other embodiments, the Cas nuclease may be from a Type-I CRISPR/Cassystem. In some embodiments, the Cas nuclease may be a component of theCascade complex of a Type-I CRISPR/Cas system. In some embodiments, theCas nuclease may be a Cas3 protein. In some embodiments, the Casnuclease may be from a Type-III CRISPR/Cas system. In some embodiments,the Cas nuclease may have an RNA cleavage activity.

In some embodiments, the RNA-guided DNA-binding agent has single-strandnickase activity, i.e., can cut one DNA strand to produce asingle-strand break, also known as a “nick.” In some embodiments, theRNA-guided DNA-binding agent comprises a Cas nickase. A nickase is anenzyme that creates a nick in dsDNA, i.e., cuts one strand but not theother of the DNA double helix. In some embodiments, a Cas nickase is aversion of a Cas nuclease (e.g., a Cas nuclease discussed above) inwhich an endonucleolytic active site is inactivated, e.g., by one ormore alterations (e.g., point mutations) in a catalytic domain. See,e.g., U.S. Pat. No. 8,889,356 for discussion of Cas nickases andexemplary catalytic domain alterations. In some embodiments, a Casnickase such as a Cas9 nickase has an inactivated RuvC or HNH domain. Anexemplary Cas9 nickase amino acid sequence is provided as SEQ ID NO:206. An exemplary Cas9 nickase mRNA ORF sequence, which includes startand stop codons, is provided as SEQ ID NO: 207. An exemplary Cas9nickase mRNA coding sequence, suitable for inclusion in a fusionprotein, is provided as SEQ ID NO: 211.

In some embodiments, the RNA-guided DNA-binding agent is modified tocontain only one functional nuclease domain. For example, the agentprotein may be modified such that one of the nuclease domains is mutatedor fully or partially deleted to reduce its nucleic acid cleavageactivity. In some embodiments, a nickase is used having a RuvC domainwith reduced activity. In some embodiments, a nickase is used having aninactive RuvC domain. In some embodiments, a nickase is used having anHNH domain with reduced activity. In some embodiments, a nickase is usedhaving an inactive HNH domain.

In some embodiments, a conserved amino acid within a Cas proteinnuclease domain is substituted to reduce or alter nuclease activity. Insome embodiments, a Cas nuclease may comprise an amino acid substitutionin the RuvC or RuvC-like nuclease domain. Exemplary amino acidsubstitutions in the RuvC or RuvC-like nuclease domain include D10A(based on the S. pyogenes Cas9 protein). See, e.g., Zetsche et al.(2015) Cell October 22:163(3): 759-771. In some embodiments, the Casnuclease may comprise an amino acid substitution in the HNH or HNH-likenuclease domain. Exemplary amino acid substitutions in the HNH orHNH-like nuclease domain include E762A, H840A, N863A, H983A, and D986A(based on the S. pyogenes Cas9 protein). See, e.g., Zetsche et al.(2015). Further exemplary amino acid substitutions include D917A,E1006A, and D1255A (based on the Francisella novicida U112 Cpf1 (FnCpf1)sequence (UniProtKB—A0Q7Q2 (CPF1_FRATN)).

In some embodiments, an mRNA encoding a nickase is provided incombination with a pair of guide RNAs that are complementary to thesense and antisense strands of the target sequence, respectively. Inthis embodiment, the guide RNAs direct the nickase to a target sequenceand introduce a DSB by generating a nick on opposite strands of thetarget sequence (i.e., double nicking). In some embodiments, use ofdouble nicking may improve specificity and reduce off-target effects. Insome embodiments, a nickase is used together with two separate guideRNAs targeting opposite strands of DNA to produce a double nick in thetarget DNA. In some embodiments, a nickase is used together with twoseparate guide RNAs that are selected to be in close proximity toproduce a double nick in the target DNA.

In some embodiments, the RNA-guided DNA-binding agent lacks cleavase andnickase activity. In some embodiments, the RNA-guided DNA-binding agentcomprises a dCas DNA-binding polypeptide. A dCas polypeptide hasDNA-binding activity while essentially lacking catalytic(cleavase/nickase) activity. In some embodiments, the dCas polypeptideis a dCas9 polypeptide. In some embodiments, the RNA-guided DNA-bindingagent lacking cleavase and nickase activity or the dCas DNA-bindingpolypeptide is a version of a Cas nuclease (e.g., a Cas nucleasediscussed above) in which its endonucleolytic active sites areinactivated, e.g., by one or more alterations (e.g., point mutations) inits catalytic domains. See, e.g., US 2014/0186958 A1; US 2015/0166980A1. An exemplary dCas9 amino acid sequence is provided as SEQ ID NO:208. An exemplary dCas9 mRNA ORF sequence, which includes start and stopcodons, is provided as SEQ ID NO: 209. An exemplary dCas9 mRNA codingsequence, suitable for inclusion in a fusion protein, is provided as SEQID NO: 212.

In some embodiments, the RNA-guided DNA-binding agent comprises one ormore heterologous functional domains (e.g., is or comprises a fusionpolypeptide).

In some embodiments, the heterologous functional domain may facilitatetransport of the RNA-guided DNA-binding agent into the nucleus of acell. For example, the heterologous functional domain may be a nuclearlocalization signal (NLS). In some embodiments, the RNA-guidedDNA-binding agent may be fused with 1-10 NLS(s). In some embodiments,the RNA-guided DNA-binding agent may be fused with 1-5 NLS(s). In someembodiments, the RNA-guided DNA-binding agent may be fused with one NLS.Where one NLS is used, the NLS may be linked at the N-terminus or theC-terminus of the RNA-guided DNA-binding agent sequence. It may also beinserted within the RNA-guided DNA binding agent sequence. In otherembodiments, the RNA-guided DNA-binding agent may be fused with morethan one NLS. In some embodiments, the RNA-guided DNA-binding agent maybe fused with 2, 3, 4, or 5 NLSs. In some embodiments, the RNA-guidedDNA-binding agent may be fused with two NLSs. In certain circumstances,the two NLSs may be the same (e.g., two SV40 NLSs) or different. In someembodiments, the RNA-guided DNA-binding agent is fused to two SV40 NLSsequences linked at the carboxy terminus. In some embodiments, theRNA-guided DNA-binding agent may be fused with two NLSs, one linked atthe N-terminus and one at the C-terminus. In some embodiments, theRNA-guided DNA-binding agent may be fused with 3 NLSs. In someembodiments, the RNA-guided DNA-binding agent may be fused with no NLS.In some embodiments, the NLS may be a monopartite sequence, such as,e.g., the SV40 NLS, PKKKRKV (SEQ ID NO: 274) or PKKKRRV (SEQ ID NO:275). In some embodiments, the NLS may be a bipartite sequence, such asthe NLS of nucleoplasmin, KRPAATKKAGQAKKKK (SEQ ID NO: 276). In aspecific embodiment, a single PKKKRKV (SEQ ID NO: 274) NLS may be linkedat the C-terminus of the RNA-guided DNA-binding agent. One or morelinkers are optionally included at the fusion site.

In some embodiments, the heterologous functional domain may be capableof modifying the intracellular half-life of the RNA-guided DNA bindingagent. In some embodiments, the half-life of the RNA-guided DNA bindingagent may be increased. In some embodiments, the half-life of theRNA-guided DNA-binding agent may be reduced. In some embodiments, theheterologous functional domain may be capable of increasing thestability of the RNA-guided DNA-binding agent. In some embodiments, theheterologous functional domain may be capable of reducing the stabilityof the RNA-guided DNA-binding agent. In some embodiments, theheterologous functional domain may act as a signal peptide for proteindegradation. In some embodiments, the protein degradation may bemediated by proteolytic enzymes, such as, for example, proteasomes,lysosomal proteases, or calpain proteases. In some embodiments, theheterologous functional domain may comprise a PEST sequence. In someembodiments, the RNA-guided DNA-binding agent may be modified byaddition of ubiquitin or a polyubiquitin chain. In some embodiments, theubiquitin may be a ubiquitin-like protein (UBL). Non-limiting examplesof ubiquitin-like proteins include small ubiquitin-like modifier (SUMO),ubiquitin cross-reactive protein (UCRP, also known asinterferon-stimulated gene-15 (ISG15)), ubiquitin-related modifier-1(URM1), neuronal-precursor-cell-expressed developmentally downregulatedprotein-8 (NEDD8, also called Rub 1 in S. cerevisiae), human leukocyteantigen F-associated (FAT10), autophagy-8 (ATG8) and -12 (ATG12), Fauubiquitin-like protein (FUB1), membrane-anchored UBL (MUB), ubiquitinfold-modifier-1 (UFM1), and ubiquitin-like protein-5 (UBLS).

In some embodiments, the heterologous functional domain may be a markerdomain. Non-limiting examples of marker domains include fluorescentproteins, purification tags, epitope tags, and reporter gene sequences.In some embodiments, the marker domain may be a fluorescent protein.Non-limiting examples of suitable fluorescent proteins include greenfluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, sfGFP, EGFP,Emerald, Azami Green, Monomeric Azami Green, CopGFP, AceGFP, ZsGreen1),yellow fluorescent proteins (e.g., YFP, EYFP, Citrine, Venus, YPet,PhiYFP, ZsYellowl), blue fluorescent proteins (e.g., EBFP, EBFP2,Azurite, mKalamal, GFPuv, Sapphire, T-sapphire), cyan fluorescentproteins (e.g., ECFP, Cerulean, CyPet, AmCyanl, Midoriishi-Cyan), redfluorescent proteins (e.g., mKate, mKate2, mPlum, DsRed monomer,mCherry, mRFP1, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem,HcRedl, AsRed2, eqFP611, mRasberry, mStrawberry, Jred), and orangefluorescent proteins (mOrange, mKO, Kusabira-Orange, MonomericKusabira-Orange, mTangerine, tdTomato) or any other suitable fluorescentprotein. In other embodiments, the marker domain may be a purificationtag and/or an epitope tag. Non-limiting exemplary tags includeglutathione-S-transferase (GST), chitin binding protein (CBP), maltosebinding protein (MBP), thioredoxin (TRX), poly(NANP), tandem affinitypurification (TAP) tag, myc, AcV5, AU1, AUS, E, ECS, E2, FLAG, HA, nus,Softag 1, Softag 3, Strep, SBP, Glu-Glu, HSV, KT3, S, S1, T7, V5, VSV-G,6×His, 8×His, biotin carboxyl carrier protein (BCCP), poly-His, andcalmodulin. Non-limiting exemplary reporter genes includeglutathione-S-transferase (GST), horseradish peroxidase (HRP),chloramphenicol acetyltransferase (CAT), beta-galactosidase,beta-glucuronidase, luciferase, or fluorescent proteins.

In additional embodiments, the heterologous functional domain may targetthe RNA-guided DNA-binding agent to a specific organelle, cell type,tissue, or organ. In some embodiments, the heterologous functionaldomain may target the RNA-guided DNA-binding agent to mitochondria.

In further embodiments, the heterologous functional domain may be aneffector domain. When the RNA-guided DNA-binding agent is directed toits target sequence, e.g., when a Cas nuclease is directed to a targetsequence by a gRNA, the effector domain may modify or affect the targetsequence. In some embodiments, the effector domain may be chosen from anucleic acid binding domain, a nuclease domain (e.g., a non-Cas nucleasedomain), an epigenetic modification domain, a transcriptional activationdomain, or a transcriptional repressor domain. In some embodiments, theheterologous functional domain is a nuclease, such as a FokI nuclease.See, e.g., U.S. Pat. No. 9,023,649. In some embodiments, theheterologous functional domain is a transcriptional activator orrepressor. See, e.g., Qi et al., “Repurposing CRISPR as an RNA-guidedplatform for sequence-specific control of gene expression,” Cell152:1173-83 (2013); Perez-Pinera et al., “RNA-guided gene activation byCRISPR-Cas9-based transcription factors,” Nat. Methods 10:973-6 (2013);Mali et al., “CAS9 transcriptional activators for target specificityscreening and paired nickases for cooperative genome engineering,” Nat.Biotechnol. 31:833-8 (2013); Gilbert et al., “CRISPR-mediated modularRNA-guided regulation of transcription in eukaryotes,” Cell 154:442-51(2013). As such, the RNA-guided DNA-binding agent essentially becomes atranscription factor that can be directed to bind a desired targetsequence using a guide RNA.

B. Modified gRNAs and mRNAs

In some embodiments, the gRNA is chemically modified. A gRNA comprisingone or more modified nucleosides or nucleotides is called a “modified”gRNA or “chemically modified” gRNA, to describe the presence of one ormore non-naturally and/or naturally occurring components orconfigurations that are used instead of or in addition to the canonicalA, G, C, and U residues. In some embodiments, a modified gRNA issynthesized with a non-canonical nucleoside or nucleotide, is herecalled “modified.” Modified nucleosides and nucleotides can include oneor more of: (i) alteration, e.g., replacement, of one or both of thenon-linking phosphate oxygens and/or of one or more of the linkingphosphate oxygens in the phosphodiester backbone linkage (an exemplarybackbone modification); (ii) alteration, e.g., replacement, of aconstituent of the ribose sugar, e.g., of the 2′ hydroxyl on the ribosesugar (an exemplary sugar modification); (iii) wholesale replacement ofthe phosphate moiety with “dephospho” linkers (an exemplary backbonemodification); (iv) modification or replacement of a naturally occurringnucleobase, including with a non-canonical nucleobase (an exemplary basemodification); (v) replacement or modification of the ribose-phosphatebackbone (an exemplary backbone modification); (vi) modification of the3′ end or 5′ end of the oligonucleotide, e.g., removal, modification orreplacement of a terminal phosphate group or conjugation of a moiety,cap or linker (such 3′ or 5′ cap modifications may comprise a sugarand/or backbone modification); and (vii) modification or replacement ofthe sugar (an exemplary sugar modification).

As noted above, in some embodiments, a composition or formulationdisclosed herein comprises an mRNA comprising an open reading frame(ORF) encoding an RNA-guided DNA binding agent, such as a Cas nucleaseas described herein. In some embodiments, an mRNA comprising an ORFencoding an RNA-guided DNA binding agent, such as a Cas nuclease, isprovided, used, or administered. In some embodiments, the ORF encodingan RNA-guided DNA nuclease is a “modified RNA-guided DNA binding agentORF” or simply a “modified ORF,” which is used as shorthand to indicatethat the ORF is modified in one or more of the following ways: (1) themodified ORF has a uridine content ranging from its minimum uridinecontent to 150% of the minimum uridine content; (2) the modified ORF hasa uridine dinucleotide content ranging from its minimum uridinedinucleotide content to 150% of the minimum uridine dinucleotidecontent; (3) the modified ORF has at least 90% identity to any one ofSEQ ID NOs: 201, 204, 210, 214, 215, 223, 224, 250, 252, 254, 265, or266; (4) the modified ORF consists of a set of codons of which at least75% of the codons are codons listed in the Table 3A of Minimal UridineCodons; or (5) the modified ORF comprises at least one modified uridine.In some embodiments, the modified ORF is modified in at least two,three, or four of the foregoing ways. In some embodiments, the modifiedORF comprises at least one modified uridine and is modified in at leastone, two, three, or all of (1)-(4) above.

TABLE 3A of Minimal Uridine Codons Amino Acid Minimal uridine codon AAlanine GCA or GCC or GCG G Glycine GGA or GGC or GGG V Valine GUC orGUA or GUG D Aspartic acid GAC E Glutamic acid GAA or GAG I IsoleucineAUC or AUA T Threonine ACA or ACC or ACG N Asparagine AAC K Lysine AAGor AAA S Serine AGC R Arginine AGA or AGG L Leucine CUG or CUA or CUC PProline CCG or CCA or CCC H Histidine CAC Q Glutamine CAG or CAA FPhenylalanine UUC Y Tyrosine UAC C Cysteine UGC W Tryptophan UGG MMethionine AUG

In any of the foregoing embodiments, the modified ORF may consist of aset of codons of which at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or100% of the codons are codons listed in Table 3A showing Minimal UridineCodons.

In any of the foregoing embodiments, the modified ORF may comprise asequence with at least 90%, 95%, 98%, 99%, or 100% identity to any oneof SEQ ID NO: 201, 204, 210, 214, 215, 223, 224, 250, 252, 254, 265, or266.

In any of the foregoing embodiments, the modified ORF may have a uridinecontent ranging from its minimum uridine content to 150%, 145%, 140%,135%, 130%, 125%, 120%, 115%, 110%, 105%, 104%, 103%, 102%, or 101% ofthe minimum uridine content.

In any of the foregoing embodiments, the modified ORF may have a uridinedinucleotide content ranging from its minimum uridine dinucleotidecontent to 150%, 145%, 140%, 135%, 130%, 125%, 120%, 115%, 110%, 105%,104%, 103%, 102%, or 101% of the minimum uridine dinucleotide content.

In any of the foregoing embodiments, the modified ORF may comprise amodified uridine at least at one, a plurality of, or all uridinepositions. In some embodiments, the modified uridine is a uridinemodified at the 5 position, e.g., with a halogen, methyl, or ethyl. Insome embodiments, the modified uridine is a pseudouridine modified atthe 1 position, e.g., with a halogen, methyl, or ethyl. The modifieduridine can be, for example, pseudouridine, N1-methyl-pseudouridine,5-methoxyuridine, 5-iodouridine, or a combination thereof. In someembodiments, the modified uridine is 5-methoxyuridine. In someembodiments, the modified uridine is 5-iodouridine. In some embodiments,the modified uridine is pseudouridine. In some embodiments, the modifieduridine is N1-methyl-pseudouridine. In some embodiments, the modifieduridine is a combination of pseudouridine and N1-methyl-pseudouridine.In some embodiments, the modified uridine is a combination ofpseudouridine and 5-methoxyuridine. In some embodiments, the modifieduridine is a combination of N1-methyl pseudouridine and5-methoxyuridine. In some embodiments, the modified uridine is acombination of 5-iodouridine and N1-methyl-pseudouridine. In someembodiments, the modified uridine is a combination of pseudouridine and5-iodouridine. In some embodiments, the modified uridine is acombination of 5-iodouridine and 5-methoxyuridine.

In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% ofthe uridine positions in an mRNA according to the disclosure aremodified uridines. In some embodiments, 10%-25%, 15-25%, 25-35%, 35-45%,45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of the uridinepositions in an mRNA according to the disclosure are modified uridines,e.g., 5-methoxyuridine, 5-iodouridine, N1-methyl pseudouridine,pseudouridine, or a combination thereof. In some embodiments, 10%-25%,15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or90-100% of the uridine positions in an mRNA according to the disclosureare 5-methoxyuridine. In some embodiments, 10%-25%, 15-25%, 25-35%,35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of theuridine positions in an mRNA according to the disclosure arepseudouridine. In some embodiments, 10%-25%, 15-25%, 25-35%, 35-45%,45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of the uridinepositions in an mRNA according to the disclosure are N1-methylpseudouridine. In some embodiments, 10%-25%, 15-25%, 25-35%, 35-45%,45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of the uridinepositions in an mRNA according to the disclosure are 5-iodouridine. Insome embodiments, 10%-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%,65-75%, 75-85%, 85-95%, or 90-100% of the uridine positions in an mRNAaccording to the disclosure are 5-methoxyuridine, and the remainder areN1-methyl pseudouridine. In some embodiments, 10%-25%, 15-25%, 25-35%,35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of theuridine positions in an mRNA according to the disclosure are5-iodouridine, and the remainder are N1-methyl pseudouridine.

In some embodiments, the mRNA comprises at least one UTR from anexpressed mammalian mRNA, such as a constitutively expressed mRNA. AnmRNA is considered constitutively expressed in a mammal if it iscontinually transcribed in at least one tissue of a healthy adultmammal. In some embodiments, the mRNA comprises a 5′ UTR, 3′ UTR, or 5′and 3′ UTRs from an expressed mammalian RNA, such as a constitutivelyexpressed mammalian mRNA. Actin mRNA is an example of a constitutivelyexpressed mRNA.

In some embodiments, the mRNA comprises at least one UTR fromHydroxysteroid 17-Beta Dehydrogenase 4 (HSD17B4 or HSD), e.g., a 5′ UTRfrom HSD. In some embodiments, the mRNA comprises at least one UTR froma globin mRNA, for example, human alpha globin (HBA) mRNA, human betaglobin (HBB) mRNA, or Xenopus laevis beta globin (XBG) mRNA. In someembodiments, the mRNA comprises a 5′ UTR, 3′ UTR, or 5′ and 3′ UTRs froma globin mRNA, such as HBA, HBB, or XBG. In some embodiments, the mRNAcomprises a 5′ UTR from bovine growth hormone, cytomegalovirus (CMV),mouse Hba-a1, HSD, an albumin gene, HBA, HBB, or XBG. In someembodiments, the mRNA comprises a 3′ UTR from bovine growth hormone,cytomegalovirus, mouse Hba-a1, HSD, an albumin gene, HBA, HBB, or XBG.In some embodiments, the mRNA comprises 5′ and 3′ UTRs from bovinegrowth hormone, cytomegalovirus, mouse Hba-a1, HSD, an albumin gene,HBA, HBB, XBG, heat shock protein 90 (Hsp90), glyceraldehyde 3-phosphatedehydrogenase (GAPDH), beta-actin, alpha-tubulin, tumor protein (p53),or epidermal growth factor receptor (EGFR).

In some embodiments, the mRNA comprises 5′ and 3′ UTRs that are from thesame source, e.g., a constitutively expressed mRNA such as actin,albumin, or a globin such as HBA, HBB, or XBG.

In some embodiments, the mRNA does not comprise a 5′ UTR, e.g., thereare no additional nucleotides between the 5′ cap and the start codon. Insome embodiments, the mRNA comprises a Kozak sequence (described below)between the 5′ cap and the start codon, but does not have any additional5′ UTR. In some embodiments, the mRNA does not comprise a 3′ UTR, e.g.,there are no additional nucleotides between the stop codon and thepoly-A tail.

In some embodiments, the mRNA comprises a Kozak sequence. The Kozaksequence can affect translation initiation and the overall yield of apolypeptide translated from an mRNA. A Kozak sequence includes amethionine codon that can function as the start codon. A minimal Kozaksequence is NNNRUGN wherein at least one of the following is true: thefirst N is A or G and the second N is G. In the context of a nucleotidesequence, R means a purine (A or G). In some embodiments, the Kozaksequence is RNNRUGN, NNNRUGG, RNNRUGG, RNNAUGN, NNNAUGG, or RNNAUGG. Insome embodiments, the Kozak sequence is rccRUGg with zero mismatches orwith up to one or two mismatches to positions in lowercase. In someembodiments, the Kozak sequence is rccAUGg with zero mismatches or withup to one or two mismatches to positions in lowercase. In someembodiments, the Kozak sequence is gccRccAUGG (SEQ ID NO: 277) with zeromismatches or with up to one, two, or three mismatches to positions inlowercase. In some embodiments, the Kozak sequence is gccAccAUG withzero mismatches or with up to one, two, three, or four mismatches topositions in lowercase. In some embodiments, the Kozak sequence isGCCACCAUG. In some embodiments, the Kozak sequence is gccgccRccAUGG (SEQID NO: 278) with zero mismatches or with up to one, two, three, or fourmismatches to positions in lowercase.

In some embodiments, the mRNA comprising an ORF encoding an RNA-guidedDNA binding agent comprises a sequence having at least 90% identity toSEQ ID NO: 1, optionally wherein the ORF of SEQ ID NO: 1 (i.e., SEQ IDNO: 204) is substituted with an alternative ORF of any one of SEQ ID NO:210, 214, 215, 223, 224, 250, 252, 254, 265, or 266.

In some embodiments, the mRNA comprising an ORF encoding an RNA-guidedDNA binding agent comprises a sequence having at least 90% identity toSEQ ID NO: 244, optionally wherein the ORF of SEQ ID NO: 244 (i.e., SEQID NO: 204) is substituted with an alternative ORF of any one of SEQ IDNO: 210, 214, 215, 223, 224, 250, 252, 254, 265, or 266.

In some embodiments, the mRNA comprising an ORF encoding an RNA-guidedDNA binding agent comprises a sequence having at least 90% identity toSEQ ID NO: 256, optionally wherein the ORF of SEQ ID NO: 256 (i.e., SEQID NO: 204) is substituted with an alternative ORF of any one of SEQ IDNO: 210, 214, 215, 223, 224, 250, 252, 254, 265, or 266.

In some embodiments, the mRNA comprising an ORF encoding an RNA-guidedDNA binding agent comprises a sequence having at least 90% identity toSEQ ID NO: 257, optionally wherein the ORF of SEQ ID NO: 257 (i.e., SEQID NO: 204) is substituted with an alternative ORF of any one of SEQ IDNO: 210, 214, 215, 223, 224, 250, 252, 254, 265, or 266.

In some embodiments, the mRNA comprising an ORF encoding an RNA-guidedDNA binding agent comprises a sequence having at least 90% identity toSEQ ID NO: 257, optionally wherein the ORF of SEQ ID NO: 258 (i.e., SEQID NO: 204) is substituted with an alternative ORF of any one of SEQ IDNO: 210, 214, 215, 223, 224, 250, 252, 254, 265, or 266.

In some embodiments, the mRNA comprising an ORF encoding an RNA-guidedDNA binding agent comprises a sequence having at least 90% identity toSEQ ID NO: 259, optionally wherein the ORF of SEQ ID NO: 259 (i.e., SEQID NO: 204) is substituted with an alternative ORF of any one of SEQ IDNO: 210, 214, 215, 223, 224, 250, 252, 254, 265, or 266.

In some embodiments, the mRNA comprising an ORF encoding an RNA-guidedDNA binding agent comprises a sequence having at least 90% identity toSEQ ID NO: 260, optionally wherein the ORF of SEQ ID NO: 260 (i.e., SEQID NO: 204) is substituted with an alternative ORF of any one of SEQ IDNO: 210, 214, 215, 223, 224, 250, 252, 254, 265, or 266.

In some embodiments, the mRNA comprising an ORF encoding an RNA-guidedDNA binding agent comprises a sequence having at least 90% identity toSEQ ID NO: 261, optionally wherein the ORF of SEQ ID NO: 261 (i.e., SEQID NO: 204) is substituted with an alternative ORF of any one of SEQ IDNO: 210, 214, 215, 223, 224, 250, 252, 254, 265, or 266.

In some embodiments, the degree of identity to the optionallysubstituted sequences of SEQ ID NOs 243, 244, or 256-261 is 95%. In someembodiments, the degree of identity to the optionally substitutedsequences of SEQ ID NOs 243, 244, or 256-261 is 98%. In someembodiments, the degree of identity to the optionally substitutedsequences of SEQ ID NOs 243, 244, or 256-261 is 99%. In someembodiments, the degree of identity to the optionally substitutedsequences of SEQ ID NOs 243, 244, or 256-261 is 100%.

In some embodiments, an mRNA disclosed herein comprises a 5′ cap, suchas a Cap0, Cap1, or Cap2. A 5′ cap is generally a 7-methylguanineribonucleotide (which may be further modified, as discussed below e.g.with respect to ARCA) linked through a 5′-triphosphate to the 5′position of the first nucleotide of the 5′-to-3′ chain of the mRNA,i.e., the first cap-proximal nucleotide. In Cap0, the riboses of thefirst and second cap-proximal nucleotides of the mRNA both comprise a2′-hydroxyl. In Cap1, the riboses of the first and second transcribednucleotides of the mRNA comprise a 2′-methoxy and a 2′-hydroxyl,respectively. In Cap2, the riboses of the first and second cap-proximalnucleotides of the mRNA both comprise a 2′-methoxy. See, e.g., Katibahet al. (2014) Proc Natl Acad Sci USA 111(33):12025-30; Abbas et al.(2017) Proc Natl Acad Sci USA 114(11):E2106-E2115. Most endogenoushigher eukaryotic mRNAs, including mammalian mRNAs such as human mRNAs,comprise Cap1 or Cap2. Cap0 and other cap structures differing from Cap1and Cap2 may be immunogenic in mammals, such as humans, due torecognition as “non-self” by components of the innate immune system suchas IFIT-1 and IFIT-5, which can result in elevated cytokine levelsincluding type I interferon. Components of the innate immune system suchas IFIT-1 and IFIT-5 may also compete with eIF4E for binding of an mRNAwith a cap other than Cap1 or Cap2, potentially inhibiting translationof the mRNA.

A cap can be included co-transcriptionally. For example, ARCA(anti-reverse cap analog; Thermo Fisher Scientific Cat. No. AM8045) is acap analog comprising a 7-methylguanine 3′-methoxy-5′-triphosphatelinked to the 5′ position of a guanine ribonucleotide which can beincorporated in vitro into a transcript at initiation. ARCA results in aCap0 cap in which the 2′ position of the first cap-proximal nucleotideis hydroxyl. See, e.g., Stepinski et al., (2001) “Synthesis andproperties of mRNAs containing the novel ‘anti-reverse’ cap analogs7-methyl(3′-O-methyl)GpppG and 7-methyl(3′deoxy)GpppG,” RNA 7:1486-1495. The ARCA structure is shown below.

CleanCap™ AG (m7G(5′)ppp(5′)(2′OMeA)pG; TriLink Biotechnologies Cat. No.N-7113) or CleanCap™ GG (m7G(5′)ppp(5′)(2′OMeG)pG; TriLinkBiotechnologies Cat. No. N-7133) can be used to provide a Cap1 structureco-transcriptionally. 3′-O-methylated versions of CleanCap™ AG andCleanCap™ GG are also available from TriLink Biotechnologies as Cat.Nos. N-7413 and N-7433, respectively. The CleanCap™ AG structure isshown below.

Alternatively, a cap can be added to an RNA post-transcriptionally. Forexample, Vaccinia capping enzyme is commercially available (New EnglandBiolabs Cat. No. M2080S) and has RNA triphosphatase andguanylyltransferase activities, provided by its D1 subunit, and guaninemethyltransferase, provided by its D12 subunit. As such, it can add a7-methylguanine to an RNA, so as to give Cap0, in the presence ofS-adenosyl methionine and GTP. See, e.g., Guo, P. and Moss, B. (1990)Proc. Natl. Acad. Sci. USA 87, 4023-4027; Mao, X. and Shuman, S. (1994)J. Biol. Chem. 269, 24472-24479. For additional discussion of caps andcapping approaches, see, e.g., WO2017/053297 and Ishikawa et al., Nucl.Acids. Symp. Ser. (2009) No. 53, 129-130.

In some embodiments, the mRNA further comprises a poly-adenylated(poly-A) tail. In some embodiments, the poly-A tail comprises at least20, 30, 40, 50, 60, 70, 80, 90, or 100 adenines, optionally up to 300adenines. In some embodiments, the poly-A tail comprises 95, 96, 97, 98,99, or 100 adenine nucleotides. In some instances, the poly-A tail is“interrupted” with one or more non-adenine nucleotide “anchors” at oneor more locations within the poly-A tail. The poly-A tails may compriseat least 8 consecutive adenine nucleotides, but also comprise one ormore non-adenine nucleotide. As used herein, “non-adenine nucleotides”refer to any natural or non-natural nucleotides that do not compriseadenine. Guanine, thymine, and cytosine nucleotides are exemplarynon-adenine nucleotides. Thus, the poly-A tails on the mRNA describedherein may comprise consecutive adenine nucleotides located 3′ tonucleotides encoding an RNA-guided DNA binding agent or a sequence ofinterest. In some instances, the poly-A tails on mRNA comprisenon-consecutive adenine nucleotides located 3′ to nucleotides encodingan RNA-guided DNA binding agent or a sequence of interest, whereinnon-adenine nucleotides interrupt the adenine nucleotides at regular orirregularly spaced intervals.

In some embodiments, the one or more non-adenine nucleotides arepositioned to interrupt the consecutive adenine nucleotides so that apoly(A) binding protein can bind to a stretch of consecutive adeninenucleotides. In some embodiments, one or more non-adenine nucleotide(s)is located after at least 8, 9, 10, 11, or 12 consecutive adeninenucleotides. In some embodiments, the one or more non-adenine nucleotideis located after at least 8-50 consecutive adenine nucleotides. In someembodiments, the one or more non-adenine nucleotide is located after atleast 8-100 consecutive adenine nucleotides. In some embodiments, thenon-adenine nucleotide is after one, two, three, four, five, six, orseven adenine nucleotides and is followed by at least 8 consecutiveadenine nucleotides.

The poly-A tail may comprise one sequence of consecutive adeninenucleotides followed by one or more non-adenine nucleotides, optionallyfollowed by additional adenine nucleotides.

In some embodiments, the poly-A tail comprises or contains onenon-adenine nucleotide or one consecutive stretch of 2-10 non-adeninenucleotides. In some embodiments, the non-adenine nucleotide(s) islocated after at least 8, 9, 10, 11, or 12 consecutive adeninenucleotides. In some instances, the one or more non-adenine nucleotidesare located after at least 8-50 consecutive adenine nucleotides. In someembodiments, the one or more non-adenine nucleotides are located afterat least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, or 50 consecutive adenine nucleotides.

In some embodiments, the non-adenine nucleotide is guanine, cytosine, orthymine. In some instances, the non-adenine nucleotide is a guaninenucleotide. In some embodiments, the non-adenine nucleotide is acytosine nucleotide. In some embodiments, the non-adenine nucleotide isa thymine nucleotide. In some instances, where more than one non-adeninenucleotide is present, the non-adenine nucleotide may be selected from:a) guanine and thymine nucleotides; b) guanine and cytosine nucleotides;c) thymine and cytosine nucleotides; or d) guanine, thymine and cytosinenucleotides. An exemplary poly-A tail comprising non-adenine nucleotidesis provided as SEQ ID NO: 4.

In some embodiments, the mRNA further comprises a poly-adenylated(poly-A) tail. In some instances, the poly-A tail is “interrupted” withone or more non-adenine nucleotide “anchors” at one or more locationswithin the poly-A tail. The poly-A tails may comprise at least 8consecutive adenine nucleotides, but also comprise one or morenon-adenine nucleotide. As used herein, “non-adenine nucleotides” referto any natural or non-natural nucleotides that do not comprise adenine.Guanine, thymine, and cytosine nucleotides are exemplary non-adeninenucleotides. Thus, the poly-A tails on the mRNA described herein maycomprise consecutive adenine nucleotides located 3′ to nucleotidesencoding an RNA-guided DNA-binding agent or a sequence of interest. Insome instances, the poly-A tails on mRNA comprise non-consecutiveadenine nucleotides located 3′ to nucleotides encoding an RNA-guidedDNA-binding agent or a sequence of interest, wherein non-adeninenucleotides interrupt the adenine nucleotides at regular or irregularlyspaced intervals.

In some embodiments, the one or more non-adenine nucleotides arepositioned to interrupt the consecutive adenine nucleotides so that apoly(A) binding protein can bind to a stretch of consecutive adeninenucleotides. In some embodiments, one or more non-adenine nucleotide(s)is located after at least 8, 9, 10, 11, or 12 consecutive adeninenucleotides. In some embodiments, the one or more non-adenine nucleotideis located after at least 8-50 consecutive adenine nucleotides. In someembodiments, the one or more non-adenine nucleotide is located after atleast 8-100 consecutive adenine nucleotides. In some embodiments, thenon-adenine nucleotide is after one, two, three, four, five, six, orseven adenine nucleotides and is followed by at least 8 consecutiveadenine nucleotides.

The poly-A tail of the present invention may comprise one sequence ofconsecutive adenine nucleotides followed by one or more non-adeninenucleotides, optionally followed by additional adenine nucleotides.

In some embodiments, the poly-A tail comprises or contains onenon-adenine nucleotide or one consecutive stretch of 2-10 non-adeninenucleotides. In some embodiments, the non-adenine nucleotide(s) islocated after at least 8, 9, 10, 11, or 12 consecutive adeninenucleotides. In some instances, the one or more non-adenine nucleotidesare located after at least 8-50 consecutive adenine nucleotides. In someembodiments, the one or more non-adenine nucleotides are located afterat least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, or 50 consecutive adenine nucleotides.

In some embodiments, the non-adenine nucleotide is guanine, cytosine, orthymine. In some instances, the non-adenine nucleotide is a guaninenucleotide. In some embodiments, the non-adenine nucleotide is acytosine nucleotide. In some embodiments, the non-adenine nucleotide isa thymine nucleotide. In some instances, where more than one non-adeninenucleotide is present, the non-adenine nucleotide may be selected from:a) guanine and thymine nucleotides; b) guanine and cytosine nucleotides;c) thymine and cytosine nucleotides; or d) guanine, thymine and cytosinenucleotides. An exemplary poly-A tail comprising non-adenine nucleotidesis provided as SEQ ID NO: 4:

AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACCGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAA.

Chemical modifications such as those listed above can be combined toprovide modified gRNAs and/or mRNAs comprising nucleosides andnucleotides (collectively “residues”) that can have two, three, four, ormore modifications. For example, a modified residue can have a modifiedsugar and a modified nucleobase. In some embodiments, every base of agRNA is modified, e.g., all bases have a modified phosphate group, suchas a phosphorothioate group. In certain embodiments, all, orsubstantially all, of the phosphate groups of an gRNA molecule arereplaced with phosphorothioate groups. In some embodiments, modifiedgRNAs comprise at least one modified residue at or near the 5′ end ofthe RNA. In some embodiments, modified gRNAs comprise at least onemodified residue at or near the 3′ end of the RNA.

In some embodiments, the gRNA comprises one, two, three or more modifiedresidues. In some embodiments, at least 5% (e.g., at least 5%, at least10%, at least 15%, at least 20%, at least 25%, at least 30%, at least35%, at least 40%, at least 45%, at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, or 100%) of the positions in a modifiedgRNA are modified nucleosides or nucleotides.

Unmodified nucleic acids can be prone to degradation by, e.g.,intracellular nucleases or those found in serum. For example, nucleasescan hydrolyze nucleic acid phosphodiester bonds. Accordingly, in oneaspect the gRNAs described herein can contain one or more modifiednucleosides or nucleotides, e.g., to introduce stability towardintracellular or serum-based nucleases. In some embodiments, themodified gRNA molecules described herein can exhibit a reduced innateimmune response when introduced into a population of cells, both in vivoand ex vivo. The term “innate immune response” includes a cellularresponse to exogenous nucleic acids, including single stranded nucleicacids, which involves the induction of cytokine expression and release,particularly the interferons, and cell death.

In some embodiments of a backbone modification, the phosphate group of amodified residue can be modified by replacing one or more of the oxygenswith a different substituent. Further, the modified residue, e.g.,modified residue present in a modified nucleic acid, can include thewholesale replacement of an unmodified phosphate moiety with a modifiedphosphate group as described herein. In some embodiments, the backbonemodification of the phosphate backbone can include alterations thatresult in either an uncharged linker or a charged linker withunsymmetrical charge distribution.

Examples of modified phosphate groups include, phosphorothioate,phosphoroselenates, borano phosphates, borano phosphate esters, hydrogenphosphonates, phosphoroamidates, alkyl or aryl phosphonates andphosphotriesters. The phosphorous atom in an unmodified phosphate groupis achiral. However, replacement of one of the non-bridging oxygens withone of the above atoms or groups of atoms can render the phosphorousatom chiral. The stereogenic phosphorous atom can possess either the “R”configuration (herein Rp) or the “S” configuration (herein Sp). Thebackbone can also be modified by replacement of a bridging oxygen,(i.e., the oxygen that links the phosphate to the nucleoside), withnitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates)and carbon (bridged methylenephosphonates). The replacement can occur ateither linking oxygen or at both of the linking oxygens.

The phosphate group can be replaced by non-phosphorus containingconnectors in certain backbone modifications. In some embodiments, thecharged phosphate group can be replaced by a neutral moiety. Examples ofmoieties which can replace the phosphate group can include, withoutlimitation, e.g., methyl phosphonate, hydroxylamino, siloxane,carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxidelinker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime,methyleneimino, methylenemethylimino, methylenehydrazo,methylenedimethylhydrazo and methyleneoxymethylimino.

Scaffolds that can mimic nucleic acids can also be constructed whereinthe phosphate linker and ribose sugar are replaced by nuclease resistantnucleoside or nucleotide surrogates. Such modifications may comprisebackbone and sugar modifications. In some embodiments, the nucleobasescan be tethered by a surrogate backbone. Examples can include, withoutlimitation, the morpholino, cyclobutyl, pyrrolidine and peptide nucleicacid (PNA) nucleoside surrogates.

The modified nucleosides and modified nucleotides can include one ormore modifications to the sugar group, i.e. at sugar modification. Forexample, the 2′ hydroxyl group (OH) can be modified, e.g. replaced witha number of different “oxy” or “deoxy” substituents. In someembodiments, modifications to the 2′ hydroxyl group can enhance thestability of the nucleic acid since the hydroxyl can no longer bedeprotonated to form a 2′-alkoxide ion.

Examples of 2′ hydroxyl group modifications can include alkoxy oraryloxy (OR, wherein “R” can be, e.g., alkyl, cycloalkyl, aryl, aralkyl,heteroaryl or a sugar); polyethyleneglycols (PEG),O(CH₂CH₂O)_(n)CH₂CH₂OR wherein R can be, e.g., H or optionallysubstituted alkyl, and n can be an integer from 0 to 20 (e.g., from 0 to4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to 4, from 1 to 8,from 1 to 10, from 1 to 16, from 1 to 20, from 2 to 4, from 2 to 8, from2 to 10, from 2 to 16, from 2 to 20, from 4 to 8, from 4 to 10, from 4to 16, and from 4 to 20). In some embodiments, the 2′ hydroxyl groupmodification can be 2′-O-Me. In some embodiments, the 2′ hydroxyl groupmodification can be a 2′-fluoro modification, which replaces the 2′hydroxyl group with a fluoride. In some embodiments, the 2′ hydroxylgroup modification can include “locked” nucleic acids (LNA) in which the2′ hydroxyl can be connected, e.g., by a C₁₋₆ alkylene or C₁₋₆heteroalkylene bridge, to the 4′ carbon of the same ribose sugar, whereexemplary bridges can include methylene, propylene, ether, or aminobridges; O-amino (wherein amino can be, e.g., NH₂; alkylamino,dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, ordiheteroarylamino, ethylenediamine, or polyamino) and aminoalkoxy,O(CH₂)_(n)-amino, (wherein amino can be, e.g., NH₂; alkylamino,dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, ordiheteroarylamino, ethylenediamine, or polyamino). In some embodiments,the 2′ hydroxyl group modification can included “unlocked” nucleic acids(UNA) in which the ribose ring lacks the C2′-C3′ bond. In someembodiments, the 2′ hydroxyl group modification can include themethoxyethyl group (MOE), (OCH₂CH₂OCH₃, e.g., a PEG derivative).

“Deoxy” 2′ modifications can include hydrogen (i.e. deoxyribose sugars,e.g., at the overhang portions of partially dsRNA); halo (e.g., bromo,chloro, fluoro, or iodo); amino (wherein amino can be, e.g., NH₂;alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino,heteroarylamino, diheteroarylamino, or amino acid);NH(CH₂CH₂NH)_(n)CH₂CH₂— amino (wherein amino can be, e.g., as describedherein), —NHC(O)R (wherein R can be, e.g., alkyl, cycloalkyl, aryl,aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl;thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which maybe optionally substituted with e.g., an amino as described herein.

The sugar modification can comprise a sugar group which may also containone or more carbons that possess the opposite stereochemicalconfiguration than that of the corresponding carbon in ribose. Thus, amodified nucleic acid can include nucleotides containing e.g.,arabinose, as the sugar. The modified nucleic acids can also includeabasic sugars. These abasic sugars can also be further modified at oneor more of the constituent sugar atoms. The modified nucleic acids canalso include one or more sugars that are in the L form, e.g.L-nucleosides.

The modified nucleosides and modified nucleotides described herein,which can be incorporated into a modified nucleic acid, can include amodified base, also called a nucleobase. Examples of nucleobasesinclude, but are not limited to, adenine (A), guanine (G), cytosine (C),and uracil (U). These nucleobases can be modified or wholly replaced toprovide modified residues that can be incorporated into modified nucleicacids. The nucleobase of the nucleotide can be independently selectedfrom a purine, a pyrimidine, a purine analog, or pyrimidine analog. Insome embodiments, the nucleobase can include, for example,naturally-occurring and synthetic derivatives of a base.

In embodiments employing a dual guide RNA, each of the crRNA and thetracr RNA can contain modifications. Such modifications may be at one orboth ends of the crRNA and/or tracr RNA. In embodiments comprising ansgRNA, one or more residues at one or both ends of the sgRNA may bechemically modified, or the entire sgRNA may be chemically modified.Certain embodiments comprise a 5′ end modification. Certain embodimentscomprise a 3′ end modification. In certain embodiments, one or more orall of the nucleotides in single stranded overhang of a guide RNAmolecule are deoxynucleotides.

In some embodiments, the guide RNAs disclosed herein comprise one of themodification patterns disclosed in U.S. 62/431,756, filed Dec. 8, 2016,titled “Chemically Modified Guide RNAs,” the contents of which arehereby incorporated by reference in their entirety.

In some embodiments, the invention comprises a gRNA comprising one ormore modifications. In some embodiments, the modification comprises a2′-O-methyl (2′-O-Me) modified nucleotide. In some embodiments, themodification comprises a phosphorothioate (PS) bond between nucleotides.

The terms “mA,” “mC,” “mU,” or “mG” may be used to denote a nucleotidethat has been modified with 2′-O-Me.

Modification of 2′-O-methyl can be depicted as follows:

Another chemical modification that has been shown to influencenucleotide sugar rings is halogen substitution. For example, 2′-fluoro(2′-F) substitution on nucleotide sugar rings can increaseoligonucleotide binding affinity and nuclease stability.

In this application, the terms “fA,” “fC,” “fU,” or “fG” may be used todenote a nucleotide that has been substituted with 2′-F.

Substitution of 2′-F can be depicted as follows:

Phosphorothioate (PS) linkage or bond refers to a bond where a sulfur issubstituted for one nonbridging phosphate oxygen in a phosphodiesterlinkage, for example in the bonds between nucleotides bases. Whenphosphorothioates are used to generate oligonucleotides, the modifiedoligonucleotides may also be referred to as S-oligos.

A “*” may be used to depict a PS modification. In this application, theterms A*, C*, U*, or G* may be used to denote a nucleotide that islinked to the next (e.g., 3′) nucleotide with a PS bond.

In this application, the terms “mA*,” “mC*,” “mU*,” or “mG*” may be usedto denote a nucleotide that has been substituted with 2′-O-Me and thatis linked to the next (e.g., 3′) nucleotide with a PS bond.

The diagram below shows the substitution of S— into a nonbridgingphosphate oxygen, generating a PS bond in lieu of a phosphodiester bond:

Abasic nucleotides refer to those which lack nitrogenous bases. Thefigure below depicts an oligonucleotide with an abasic (also known asapurinic) site that lacks a base:

Inverted bases refer to those with linkages that are inverted from thenormal 5′ to 3′ linkage (i.e., either a 5′ to 5′ linkage or a 3′ to 3′linkage). For example:

An abasic nucleotide can be attached with an inverted linkage. Forexample, an abasic nucleotide may be attached to the terminal 5′nucleotide via a 5′ to 5′ linkage, or an abasic nucleotide may beattached to the terminal 3′ nucleotide via a 3′ to 3′ linkage. Aninverted abasic nucleotide at either the terminal 5′ or 3′ nucleotidemay also be called an inverted abasic end cap.

In some embodiments, one or more of the first three, four, or fivenucleotides at the 5′ terminus, and one or more of the last three, four,or five nucleotides at the 3′ terminus are modified. In someembodiments, the modification is a 2′-O-Me, 2′-F, inverted abasicnucleotide, PS bond, or other nucleotide modification well known in theart to increase stability and/or performance.

In some embodiments, the first four nucleotides at the 5′ terminus, andthe last four nucleotides at the 3′ terminus are linked withphosphorothioate (PS) bonds.

In some embodiments, the first three nucleotides at the 5′ terminus, andthe last three nucleotides at the 3′ terminus comprise a 2′-O-methyl(2′-O-Me) modified nucleotide. In some embodiments, the first threenucleotides at the 5′ terminus, and the last three nucleotides at the 3′terminus comprise a 2′-fluoro (2′-F) modified nucleotide. In someembodiments, the first three nucleotides at the 5′ terminus, and thelast three nucleotides at the 3′ terminus comprise an inverted abasicnucleotide.

In some embodiments, the guide RNA comprises a modified sgRNA. In someembodiments, the sgRNA comprises the modification pattern shown in SEQID No: 3, where N is any natural or non-natural nucleotide, and wherethe totality of the N's comprise a guide sequence that directs anuclease to a target sequence.

In some embodiments, the guide RNA comprises a sgRNA shown in any one ofSEQ ID No: 87-124. In some embodiments, the guide RNA comprises a sgRNAcomprising any one of the guide sequences of SEQ ID No: 5-82 and thenucleotides of SEQ ID No: 125, wherein the nucleotides of SEQ ID No: 125are on the 3′ end of the guide sequence, and wherein the guide sequencemay be modified as shown in SEQ ID No: 3.

C. Ribonucleoprotein Complex

In some embodiments, a composition is encompassed comprising one or moregRNAs comprising one or more guide sequences from Table 1 or one or moresgRNAs from Table 2 and an RNA-guided DNA binding agent, e.g., anuclease, such as a Cas nuclease, such as Cas9. In some embodiments, theencoded RNA-guided DNA-binding agent has cleavase activity, which canalso be referred to as double-strand endonuclease activity. In someembodiments, the RNA-guided DNA-binding agent comprises a Cas nuclease.Examples of Cas9 nucleases include those of the type II CRISPR systemsof S. pyogenes, S. aureus, and other prokaryotes (see, e.g., the list inthe next paragraph), and modified (e.g., engineered or mutant) versionsthereof. See, e.g., US2016/0312198 A1; US 2016/0312199 A1. Otherexamples of Cas nucleases include a Csm or Cmr complex of a type IIICRISPR system or the Cas10, Csm1, or Cmr2 subunit thereof; and a Cascadecomplex of a type I CRISPR system, or the Cas3 subunit thereof. In someembodiments, the Cas nuclease may be from a Type-IIA, Type-IIB, orType-IIC system. For discussion of various CRISPR systems and Casnucleases see, e.g., Makarova et al., NAT. REV. MICROBIOL. 9:467-477(2011); Makarova et al., NAT. REV. MICROBIOL, 13: 722-36 (2015); Shmakovet al., MOLECULAR CELL, 60:385-397 (2015).

Non-limiting exemplary species that the Cas nuclease can be derived frominclude Streptococcus pyogenes, Streptococcus thermophilus,Streptococcus sp., Staphylococcus aureus, Listeria innocua,Lactobacillus gasseri, Francisella novicida, Wolinella succinogenes,Sutterella wadsworthensis, Gammaproteobacterium, Neisseria meningitidis,Campylobacter jejuni, Pasteurella multocida, Fibrobacter succinogene,Rhodospirillum rubrum, Nocardiopsis dassonvillei, Streptomycespristinaespiralis, Streptomyces viridochromogenes, Streptomycesviridochromogenes, Streptosporangium roseum, Streptosporangium roseum,Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillusselenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii,Lactobacillus salivarius, Lactobacillus buchneri, Treponema denticola,Microscilla marina, Burkholderiales bacterium, Polaromonasnaphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothecesp., Microcystis aeruginosa, Synechococcus sp., Acetohalobiumarabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, CandidatusDesulforudis, Clostridium botulinum, Clostridium difficile, Finegoldiamagna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum,Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatiumvinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcuswatsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer,Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena,Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp.,Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotogamobilis, Thermosipho africanus, Streptococcus pasteurianus, Neisseriacinerea, Campylobacter lari, Parvibaculum lavamentivorans,Corynebacterium diphtheria, Acidaminococcus sp., Lachnospiraceaebacterium ND2006, and Acaryochloris marina.

In some embodiments, the Cas nuclease is the Cas9 nuclease fromStreptococcus pyogenes. In some embodiments, the Cas nuclease is theCas9 nuclease from Streptococcus thermophilus. In some embodiments, theCas nuclease is the Cas9 nuclease from Neisseria meningitidis. In someembodiments, the Cas nuclease is the Cas9 nuclease is fromStaphylococcus aureus. In some embodiments, the Cas nuclease is the Cpf1nuclease from Francisella novicida. In some embodiments, the Casnuclease is the Cpf1 nuclease from Acidaminococcus sp. In someembodiments, the Cas nuclease is the Cpf1 nuclease from Lachnospiraceaebacterium ND2006. In further embodiments, the Cas nuclease is the Cpf1nuclease from Francisella tularensis, Lachnospiraceae bacterium,Butyrivibrio proteoclasticus, Peregrinibacteria bacterium, Parcubacteriabacterium, Smithella, Acidaminococcus, Candidatus Methanoplasmatermitum, Eubacterium eligens, Moraxella bovoculi, Leptospira inadai,Porphyromonas crevioricanis, Prevotella disiens, or Porphyromonasmacacae. In certain embodiments, the Cas nuclease is a Cpf1 nucleasefrom an Acidaminococcus or Lachnospiraceae.

In some embodiments, the gRNA together with an RNA-guided DNA bindingagent is called a ribonucleoprotein complex (RNP). In some embodiments,the RNA-guided DNA binding agent is a Cas nuclease. In some embodiments,the gRNA together with a Cas nuclease is called a Cas RNP. In someembodiments, the RNP comprises Type-I, Type-II, or Type-III components.In some embodiments, the Cas nuclease is the Cas9 protein from theType-II CRISPR/Cas system. In some embodiment, the gRNA together withCas9 is called a Cas9 RNP.

Wild type Cas9 has two nuclease domains: RuvC and HNH. The RuvC domaincleaves the non-target DNA strand, and the HNH domain cleaves the targetstrand of DNA. In some embodiments, the Cas9 protein comprises more thanone RuvC domain and/or more than one HNH domain. In some embodiments,the Cas9 protein is a wild type Cas9. In each of the composition, use,and method embodiments, the Cas induces a double strand break in targetDNA.

Wild type Cas9 has two nuclease domains: RuvC and HNH. The RuvC domaincleaves the non-target DNA strand, and the HNH domain cleaves the targetstrand of DNA. In some embodiments, the Cas9 nuclease comprises morethan one RuvC domain and/or more than one HNH domain. In someembodiments, the Cas9 nuclease is a wild type Cas9. In some embodiments,the Cas9 is capable of inducing a double strand break in target DNA. Incertain embodiments, the Cas nuclease may cleave dsDNA, it may cleaveone strand of dsDNA, or it may not have DNA cleavase or nickaseactivity. An exemplary Cas9 amino acid sequence is provided as SEQ IDNO: 203. An exemplary Cas9 mRNA ORF sequence, which includes start andstop codons, is provided as SEQ ID NO: 204. An exemplary Cas9 mRNAcoding sequence, suitable for inclusion in a fusion protein, is providedas SEQ ID NO: 210.

In some embodiments, chimeric Cas nucleases are used, where one domainor region of the protein is replaced by a portion of a differentprotein. In some embodiments, a Cas nuclease domain may be replaced witha domain from a different nuclease such as FokI. In some embodiments, aCas nuclease may be a modified nuclease.

In other embodiments, the Cas nuclease may be from a Type-I CRISPR/Cassystem. In some embodiments, the Cas nuclease may be a component of theCascade complex of a Type-I CRISPR/Cas system. In some embodiments, theCas nuclease may be a Cas3 protein. In some embodiments, the Casnuclease may be from a Type-III CRISPR/Cas system. In some embodiments,the Cas nuclease may have an RNA cleavage activity.

In some embodiments, the RNA-guided DNA-binding agent has single-strandnickase activity, i.e., can cut one DNA strand to produce asingle-strand break, also known as a “nick.” In some embodiments, theRNA-guided DNA-binding agent comprises a Cas nickase. A nickase is anenzyme that creates a nick in dsDNA, i.e., cuts one strand but not theother of the DNA double helix. In some embodiments, a Cas nickase is aversion of a Cas nuclease (e.g., a Cas nuclease discussed above) inwhich an endonucleolytic active site is inactivated, e.g., by one ormore alterations (e.g., point mutations) in a catalytic domain. See,e.g., U.S. Pat. No. 8,889,356 for discussion of Cas nickases andexemplary catalytic domain alterations. In some embodiments, a Casnickase such as a Cas9 nickase has an inactivated RuvC or HNH domain. Anexemplary Cas9 nickase amino acid sequence is provided as SEQ ID NO:206. An exemplary Cas9 nickase mRNA ORF sequence, which includes startand stop codons, is provided as SEQ ID NO: 207. An exemplary Cas9nickase mRNA coding sequence, suitable for inclusion in a fusionprotein, is provided as SEQ ID NO: 211.

In some embodiments, the RNA-guided DNA-binding agent is modified tocontain only one functional nuclease domain. For example, the agentprotein may be modified such that one of the nuclease domains is mutatedor fully or partially deleted to reduce its nucleic acid cleavageactivity. In some embodiments, a nickase is used having a RuvC domainwith reduced activity. In some embodiments, a nickase is used having aninactive RuvC domain. In some embodiments, a nickase is used having anHNH domain with reduced activity. In some embodiments, a nickase is usedhaving an inactive HNH domain.

In some embodiments, a conserved amino acid within a Cas proteinnuclease domain is substituted to reduce or alter nuclease activity. Insome embodiments, a Cas nuclease may comprise an amino acid substitutionin the RuvC or RuvC-like nuclease domain. Exemplary amino acidsubstitutions in the RuvC or RuvC-like nuclease domain include D10A(based on the S. pyogenes Cas9 protein). See, e.g., Zetsche et al.(2015) Cell October 22:163(3): 759-771. In some embodiments, the Casnuclease may comprise an amino acid substitution in the HNH or HNH-likenuclease domain. Exemplary amino acid substitutions in the HNH orHNH-like nuclease domain include E762A, H840A, N863A, H983A, and D986A(based on the S. pyogenes Cas9 protein). See, e.g., Zetsche et al.(2015). Further exemplary amino acid substitutions include D917A,E1006A, and D1255A (based on the Francisella novicida U112 Cpf1 (FnCpf1)sequence (UniProtKB—A0Q7Q2 (CPF1_FRATN)).

In some embodiments, an mRNA encoding a nickase is provided incombination with a pair of guide RNAs that are complementary to thesense and antisense strands of the target sequence, respectively. Inthis embodiment, the guide RNAs direct the nickase to a target sequenceand introduce a DSB by generating a nick on opposite strands of thetarget sequence (i.e., double nicking). In some embodiments, use ofdouble nicking may improve specificity and reduce off-target effects. Insome embodiments, a nickase is used together with two separate guideRNAs targeting opposite strands of DNA to produce a double nick in thetarget DNA. In some embodiments, a nickase is used together with twoseparate guide RNAs that are selected to be in close proximity toproduce a double nick in the target DNA.

In some embodiments, the RNA-guided DNA-binding agent lacks cleavase andnickase activity. In some embodiments, the RNA-guided DNA-binding agentcomprises a dCas DNA-binding polypeptide. A dCas polypeptide hasDNA-binding activity while essentially lacking catalytic(cleavase/nickase) activity. In some embodiments, the dCas polypeptideis a dCas9 polypeptide. In some embodiments, the RNA-guided DNA-bindingagent lacking cleavase and nickase activity or the dCas DNA-bindingpolypeptide is a version of a Cas nuclease (e.g., a Cas nucleasediscussed above) in which its endonucleolytic active sites areinactivated, e.g., by one or more alterations (e.g., point mutations) inits catalytic domains. See, e.g., US 2014/0186958 A1; US 2015/0166980A1. An exemplary dCas9 amino acid sequence is provided as SEQ ID NO:208. An exemplary Cas9 mRNA ORF sequence, which includes start and stopcodons, is provided as SEQ ID NO: 209. An exemplary Cas9 mRNA codingsequence, suitable for inclusion in a fusion protein, is provided as SEQID NO: 212.

In some embodiments, the RNA-guided DNA-binding agent comprises one ormore heterologous functional domains (e.g., is or comprises a fusionpolypeptide).

In some embodiments, the heterologous functional domain may facilitatetransport of the RNA-guided DNA-binding agent into the nucleus of acell. For example, the heterologous functional domain may be a nuclearlocalization signal (NLS). In some embodiments, the RNA-guidedDNA-binding agent may be fused with 1-10 NLS(s). In some embodiments,the RNA-guided DNA-binding agent may be fused with 1-5 NLS(s). In someembodiments, the RNA-guided DNA-binding agent may be fused with one NLS.Where one NLS is used, the NLS may be linked at the N-terminus or theC-terminus of the RNA-guided DNA-binding agent sequence. In someembodiments, the RNA-guided DNA-binding agent may be fused C-terminallyto at least one NLS. An NLS may also be inserted within the RNA-guidedDNA binding agent sequence. In other embodiments, the RNA-guidedDNA-binding agent may be fused with more than one NLS. In someembodiments, the RNA-guided DNA-binding agent may be fused with 2, 3, 4,or 5 NLSs. In some embodiments, the RNA-guided DNA-binding agent may befused with two NLSs. In certain circumstances, the two NLSs may be thesame (e.g., two SV40 NLSs) or different. In some embodiments, theRNA-guided DNA-binding agent is fused to two SV40 NLS sequences linkedat the carboxy terminus. In some embodiments, the RNA-guided DNA-bindingagent may be fused with two NLSs, one linked at the N-terminus and oneat the C-terminus. In some embodiments, the RNA-guided DNA-binding agentmay be fused with 3 NLSs. In some embodiments, the RNA-guidedDNA-binding agent may be fused with no NLS. In some embodiments, the NLSmay be a monopartite sequence, such as, e.g., the SV40 NLS, PKKKRKV (SEQID NO: 274) or PKKKRRV (SEQ ID NO: 275). In some embodiments, the NLSmay be a bipartite sequence, such as the NLS of nucleoplasmin,KRPAATKKAGQAKKKK (SEQ ID NO: 276). In a specific embodiment, a singlePKKKRKV (SEQ ID NO: 274) NLS may be linked at the C-terminus of theRNA-guided DNA-binding agent. One or more linkers are optionallyincluded at the fusion site. In some embodiments, one or more NLS(s)according to any of the foregoing embodiments are present in theRNA-guided DNA-binding agent in combination with one or more additionalheterologous functional domains, such as any of the heterologousfunctional domains described below.

In some embodiments, the heterologous functional domain may be capableof modifying the intracellular half-life of the RNA-guided DNA bindingagent. In some embodiments, the half-life of the RNA-guided DNA bindingagent may be increased. In some embodiments, the half-life of theRNA-guided DNA-binding agent may be reduced. In some embodiments, theheterologous functional domain may be capable of increasing thestability of the RNA-guided DNA-binding agent. In some embodiments, theheterologous functional domain may be capable of reducing the stabilityof the RNA-guided DNA-binding agent. In some embodiments, theheterologous functional domain may act as a signal peptide for proteindegradation. In some embodiments, the protein degradation may bemediated by proteolytic enzymes, such as, for example, proteasomes,lysosomal proteases, or calpain proteases. In some embodiments, theheterologous functional domain may comprise a PEST sequence. In someembodiments, the RNA-guided DNA-binding agent may be modified byaddition of ubiquitin or a polyubiquitin chain. In some embodiments, theubiquitin may be a ubiquitin-like protein (UBL). Non-limiting examplesof ubiquitin-like proteins include small ubiquitin-like modifier (SUMO),ubiquitin cross-reactive protein (UCRP, also known asinterferon-stimulated gene-15 (ISG15)), ubiquitin-related modifier-1(URM1), neuronal-precursor-cell-expressed developmentally downregulatedprotein-8 (NEDD8, also called Rub 1 in S. cerevisiae), human leukocyteantigen F-associated (FAT10), autophagy-8 (ATG8) and -12 (ATG12), Fauubiquitin-like protein (FUB1), membrane-anchored UBL (MUB), ubiquitinfold-modifier-1 (UFM1), and ubiquitin-like protein-5 (UBLS).

In some embodiments, the heterologous functional domain may be a markerdomain. Non-limiting examples of marker domains include fluorescentproteins, purification tags, epitope tags, and reporter gene sequences.In some embodiments, the marker domain may be a fluorescent protein.Non-limiting examples of suitable fluorescent proteins include greenfluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, sfGFP, EGFP,Emerald, Azami Green, Monomeric Azami Green, CopGFP, AceGFP, ZsGreen1),yellow fluorescent proteins (e.g., YFP, EYFP, Citrine, Venus, YPet,PhiYFP, ZsYellowl), blue fluorescent proteins (e.g., EBFP, EBFP2,Azurite, mKalamal, GFPuv, Sapphire, T-sapphire), cyan fluorescentproteins (e.g., ECFP, Cerulean, CyPet, AmCyanl, Midoriishi-Cyan), redfluorescent proteins (e.g., mKate, mKate2, mPlum, DsRed monomer,mCherry, mRFP1, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem,HcRedl, AsRed2, eqFP611, mRasberry, mStrawberry, Jred), and orangefluorescent proteins (mOrange, mKO, Kusabira-Orange, MonomericKusabira-Orange, mTangerine, tdTomato) or any other suitable fluorescentprotein. In other embodiments, the marker domain may be a purificationtag and/or an epitope tag. Non-limiting exemplary tags includeglutathione-S-transferase (GST), chitin binding protein (CBP), maltosebinding protein (MBP), thioredoxin (TRX), poly(NANP), tandem affinitypurification (TAP) tag, myc, AcV5, AU1, AUS, E, ECS, E2, FLAG, HA, nus,Softag 1, Softag 3, Strep, SBP, Glu-Glu, HSV, KT3, S, S1, T7, V5, VSV-G,6×His, 8×His, biotin carboxyl carrier protein (BCCP), poly-His, andcalmodulin. Non-limiting exemplary reporter genes includeglutathione-S-transferase (GST), horseradish peroxidase (HRP),chloramphenicol acetyltransferase (CAT), beta-galactosidase,beta-glucuronidase, luciferase, or fluorescent proteins.

In additional embodiments, the heterologous functional domain may targetthe RNA-guided DNA-binding agent to a specific organelle, cell type,tissue, or organ. In some embodiments, the heterologous functionaldomain may target the RNA-guided DNA-binding agent to mitochondria.

In further embodiments, the heterologous functional domain may be aneffector domain. When the RNA-guided DNA-binding agent is directed toits target sequence, e.g., when a Cas nuclease is directed to a targetsequence by a gRNA, the effector domain may modify or affect the targetsequence. In some embodiments, the effector domain may be chosen from anucleic acid binding domain, a nuclease domain (e.g., a non-Cas nucleasedomain), an epigenetic modification domain, a transcriptional activationdomain, or a transcriptional repressor domain. In some embodiments, theheterologous functional domain is a nuclease, such as a FokI nuclease.See, e.g., U.S. Pat. No. 9,023,649. In some embodiments, theheterologous functional domain is a transcriptional activator orrepressor. See, e.g., Qi et al., “Repurposing CRISPR as an RNA-guidedplatform for sequence-specific control of gene expression,” Cell152:1173-83 (2013); Perez-Pinera et al., “RNA-guided gene activation byCRISPR-Cas9-based transcription factors,” Nat. Methods 10:973-6 (2013);Mali et al., “CAS9 transcriptional activators for target specificityscreening and paired nickases for cooperative genome engineering,” Nat.Biotechnol. 31:833-8 (2013); Gilbert et al., “CRISPR-mediated modularRNA-guided regulation of transcription in eukaryotes,” Cell 154:442-51(2013). As such, the RNA-guided DNA-binding agent essentially becomes atranscription factor that can be directed to bind a desired targetsequence using a guide RNA.

D. Determination of Efficacy of gRNAs

In some embodiments, the efficacy of a gRNA is determined when deliveredor expressed together with other components forming an RNP. In someembodiments, the gRNA is expressed together with an RNA-guided DNAnuclease, such as a Cas protein. In some embodiments, the gRNA isdelivered to or expressed in a cell line that already stably expressesan RNA-guided DNA nuclease, such as a Cas protein. In some embodimentsthe gRNA is delivered to a cell as part of a RNP. In some embodiments,the gRNA is delivered to a cell along with a mRNA encoding an RNA-guidedDNA nuclease, such as a Cas nuclease.

As described herein, use of an RNA-guided DNA nuclease and a guide RNAdisclosed herein can lead to double-stranded breaks in the DNA which canproduce errors in the form of insertion/deletion (indel) mutations uponrepair by cellular machinery. Many mutations due to indels alter thereading frame or introduce premature stop codons and, therefore, producea non-functional protein.

In some embodiments, the efficacy of particular gRNAs is determinedbased on in vitro models. In some embodiments, the in vitro model isHEK293 cells stably expressing Cas9 (HEK293_Cas9). In some embodiments,the in vitro model is HUH7 human hepatocarcinoma cells. In someembodiments, the in vitro model is HepG2 cells. In some embodiments, thein vitro model is primary human hepatocytes. In some embodiments, the invitro model is primary cynomolgus hepatocytes. With respect to usingprimary human hepatocytes, commercially available primary humanhepatocytes can be used to provide greater consistency betweenexperiments. In some embodiments, the number of off-target sites atwhich a deletion or insertion occurs in an in vitro model (e.g., inprimary human hepatocytes) is determined, e.g., by analyzing genomic DNAfrom primary human hepatocytes transfected in vitro with Cas9 mRNA andthe guide RNA. In some embodiments, such a determination comprisesanalyzing genomic DNA from primary human hepatocytes transfected invitro with Cas9 mRNA, the guide RNA, and a donor oligonucleotide.Exemplary procedures for such determinations are provided in the workingexamples below.

In some embodiments, the efficacy of particular gRNAs is determinedacross multiple in vitro cell models for a gRNA selection process. Insome embodiments, a cell line comparison of data with selected gRNAs isperformed. In some embodiments, cross screening in multiple cell modelsis performed.

In some embodiments, the efficacy of particular gRNAs is determinedbased on in vivo models. In some embodiments, the in vivo model is arodent model. In some embodiments, the rodent model is a mouse whichexpresses a human TTR gene, which may be a mutant human TTR gene. Insome embodiments, the in vivo model is a non-human primate, for examplecynomolgus monkey.

In some embodiments, the efficacy of a guide RNA is measured by percentediting of TTR. In some embodiments, the percent editing of TTR iscompared to the percent editing necessary to achieve knockdown of TTRprotein, e.g., in the cell culture media in the case of an in vitromodel or in serum or tissue in the case of an in vivo model.

In some embodiments, the efficacy of a guide RNA is measured by thenumber and/or frequency of indels at off-target sequences within thegenome of the target cell type. In some embodiments, efficacious guideRNAs are provided which produce indels at off target sites at very lowfrequencies (e.g., <5%) in a cell population and/or relative to thefrequency of indel creation at the target site. Thus, the disclosureprovides for guide RNAs which do not exhibit off-target indel formationin the target cell type (e.g., a hepatocyte), or which produce afrequency of off-target indel formation of <5% in a cell populationand/or relative to the frequency of indel creation at the target site.In some embodiments, the disclosure provides guide RNAs which do notexhibit any off target indel formation in the target cell type (e.g.,hepatocyte). In some embodiments, guide RNAs are provided which produceindels at less than 5 off-target sites, e.g., as evaluated by one ormore methods described herein. In some embodiments, guide RNAs areprovided which produce indels at less than or equal to 4, 3, 2, or 1off-target site(s) e.g., as evaluated by one or more methods describedherein. In some embodiments, the off-target site(s) does not occur in aprotein coding region in the target cell (e.g., hepatocyte) genome.

In some embodiments, detecting gene editing events, such as theformation of insertion/deletion (“indel”) mutations and homologydirected repair (HDR) events in target DNA utilize linear amplificationwith a tagged primer and isolating the tagged amplification products(herein after referred to as “LAM-PCR,” or “Linear Amplification (LA)”method).

In some embodiments, the method comprises isolating cellular DNA from acell that has been induced to have a double strand break (DSB) andoptionally that has been provided with an HDR template to repair theDSB; performing at least one cycle of linear amplification of the DNAwith a tagged primer; isolating the linear amplification products thatcomprise tag, thereby discarding any amplification product that wasamplified with a non-tagged primer; optionally further amplifying theisolated products; and analyzing the linear amplification products, orthe further amplified products, to determine the presence or absence ofan editing event such as, for example, a double strand break, aninsertion, deletion, or HDR template sequence in the target DNA. In someinstances, the editing event can be quantified. Quantification and thelike as used herein (including in the context of HDR and non-HDR editingevents such as indels) includes detecting the frequency and/or type(s)of editing events in a population.

In some embodiments, only one cycle of linear amplification isconducted.

In some instances, the tagged primer comprises a molecular barcode. Insome embodiments, the tagged primer comprises a molecular barcode, andonly one cycle of linear amplification is conducted.

In some embodiments, the analyzing step comprises sequencing the linearamplified products or the further amplified products. Sequencing maycomprise any method known to those of skill in the art, including, nextgeneration sequencing, and cloning the linear amplification products orfurther amplified products into a plasmid and sequencing the plasmid ora portion of the plasmid. In other aspects, the analyzing step comprisesperforming digital PCR (dPCR) or droplet digital PCR (ddPCR) on thelinear amplified products or the further amplified products. In otherinstances, the analyzing step comprises contacting the linear amplifiedproducts or the further amplified products with a nucleic acid probedesigned to identify DNA comprising HDR template sequence and detectingthe probes that have bound to the linear amplified product(s) or furtheramplified product(s). In some embodiments, the method further comprisesdetermining the location of the HDR template in the target DNA.

In certain embodiments, the method further comprises determining thesequence of an insertion site in the target DNA, wherein the insertionsite is the location where the HDR template incorporates into the targetDNA, and wherein the insertion site may include some target DNA sequenceand some HDR template sequence.

In some embodiments, the linear amplification of the target DNA with atagged primer is performed for 1-50 cycles, 1-60 cycles, 1-70 cycles,1-80 cycles, 1-90 cycles, or 1-100 cycles.

In some embodiments, the linear amplification of the target DNA with atagged primer comprises a denaturation step to separate DNA duplexes, anannealing step to allow primer binding, and an elongation step. In someembodiments, the linear amplification is isothermal (does not require achange in temperature). In some embodiments, the isothermal linearamplification is a loop-mediated isothermal amplification (LAMP), astrand displacement amplification (SDA), a helicase-dependentamplification, or a nicking enzyme amplification reaction.

In some embodiments, the tagged primer anneals to the target DNA atleast 50, at least 60, at least 70, at least 80, at least 90, at least100, at least 110, at least 120, at least 130, at least 140, at least150, at least 160, at least 170, at least 180, at least 190, at least200, at least 210, at least 220, at least 230, at least 240, at least250, at least 260, at least 270, at least 280, at least 290, at least300, at least 1,000, at least 5,000, or at least 10,000 nucleotides awayfrom of the expected editing event location, e.g., the insertion,deletion, or template insertion site.

In some embodiments, the tagged primer comprises a molecular barcode. Insome embodiments, the molecular barcode comprises a sequence that is notcomplementary to the target DNA. In some embodiments, the molecularbarcode comprises 6, 8, 10, or 12 nucleotides.

In some embodiments, the tag on the primer is biotin, streptavidin,digoxigenin, a DNA sequence, or fluorescein isothiocyanate (FITC).

In some embodiments, the linear amplification product(s) are isolatedusing a capture reagent specific for the tag on the primer. In someembodiments, the capture reagent is on a bead, solid support, matrix, orcolumn. In some embodiments, the isolation step comprises contacting thelinear amplification product(s) with a capture reagent specific for thetag on the primer. In some embodiments, the capture reagent is biotin,streptavidin, digoxigenin, a DNA sequence, or fluorescein isothiocyanate(FITC).

In some embodiments, the tag is biotin and capture reagent isstreptavidin. In some embodiments, the tag is streptavidin and thecapture reagent is biotin. In some embodiments, the tag is on the 5′terminus of the primer, the 3′ terminus of the primer, or internal tothe primer. In some embodiments, the tag and/or the capture reagent isremoved after the isolation step. In some embodiments, the tag and/orthe capture reagent is not removed, and the further amplifying andanalyzing steps are performed in the presence of tag and/or capture.

In some embodiments, the further amplification is non-linear. In someembodiments, the further amplification is digital PCR, qPCR, or RT-PCR.In some embodiments, the sequencing is next generation sequencing (NGS).

In some embodiments, the target DNA is genomic or mitochondrial. In someembodiments, the target DNA is genomic DNA of a prokaryotic oreukaryotic cell. In some embodiments, the target DNA is mammalian. Thetarget DNA may be from a non-dividing cell or a dividing cell. In someembodiments, the target DNA may be from a primary cell. In someembodiments, the target DNA is from a replicating cell.

In some instances, the cellular DNA is sheared prior to linearamplification. In some embodiments, the sheared DNA has an average sizebetween 0.5 kb and 20 kb. In some instances, the cellular DNA is shearedto an average size of 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0, 2.25, 2.5,2.75, 3.0, 3.25, 3.5, 3.75, 4.0, 4.25, 4.5, 4.75, 5.0, 5.25, 5.5, 5.75,6.0, 6.25, 6.5, 6.75, 7.0, 7.25, 7.5, 7.75, 8.0, 8.25, 8.5, 8.75, 9.0,9.25, 9.5, 9.75, 10.0, 10.25, 10.5, 10.75, 11.0, 11.25, 11.5, 11.75,12.0, 12.25, 12.5, 12.75, 13.0, 13.25, 13.5, 13.75, 14.0, 14.25, 14.5,14.75, 15.0, 15.25, 15.5, 15.75, 16.0, 16.25, 16.5, 16.75, 17.0, 17.25,17.5, 17.75, 18.0, 18.25, 18.5, 18.75, 19.0, 19.25, 19.5, 19.75, or 20.0kb. In some instances, the cellular DNA is sheared to an average size ofabout 1.5 kb.

In some embodiments, the efficacy of a guide RNA is measured bysecretion of TTR. In some embodiments, secretion of TTR is measuredusing an enzyme-linked immunosorbent assay (ELISA) assay with cellculture media or serum. In some embodiments, secretion of TTR ismeasured in the same in vitro or in vivo systems or models used tomeasure editing. In some embodiments, secretion of TTR is measured inprimary human hepatocytes. In some embodiments, secretion of TTR ismeasured in HUH7 cells. In some embodiments, secretion of TTR ismeasured in HepG2 cells.

ELISA assays are generally known to the skilled artisan and can bedesigned to determine serum TTR levels. In one exemplary embodiment,blood is collected and the serum is isolated. The total TTR serum levelsmay be determined using a Mouse Prealbumin (Transthyretin) ELISA Kit(Aviva Systems Biology, Cat. OKIA00111) or similar kit for measuringhuman TTR. If no kit is available, an ELISA can be developed usingplates that are pre-coated with capture antibody specific for the TTRone is measuring. The plate is next incubated at room temperature for aperiod of time before washing. Enzyme-anti-TTR antibody conjugate isadded and inncubated. Unbound antibody conjugate is removed and theplate washed before the addition of the chromogenic substrate solutionthat reactes with the enzyme. The plate is read on an appropriate platereader at an absorbance specific for the enzyme and substrate used.

In some embodiments, the amount of TTR in cells (including those fromtissue) measures efficacy of a gRNA. In some embodiments, the amount ofTTR in cells is measured using western blot. In some embodiments, thecell used is HUH7 cells. In some embodiments, the cell used is a primaryhuman hepatocyte. In some embodiments, the cell used is a primar cellobtained from an animal. In some embodiments, the amount of TTR iscompared to the amount of glyceraldehyde 3-phosphate dehydrogenase GAPDH(a housekeeping gene) to control for changes in cell number.

III. LNP Formulations and Treatment of ATTR

In some embodiments, a method of inducing a double-stranded break (DSB)within the TTR gene is provided comprising administering a compositioncomprising a guide RNA comprising any one or more guide sequences of SEQID Nos: 5-82, or any one or more of the sgRNAs of SEQ ID Nos: 87-124. Insome embodiments, gRNAs comprising any one or more of the guidesequences of SEQ ID Nos: 5-82 are administered to induce a DSB in theTTR gene. The guide RNAs may be administered together with an RNA-guidedDNA nuclease such as a Cas nuclease (e.g., Cas9) or an mRNA or vectorencoding an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9).

In some embodiments, a method of modifying the TTR gene is providedcomprising administering a composition comprising a guide RNA comprisingany one or more of the guide sequences of SEQ ID Nos: 5-82, or any oneor more of the sgRNAs of SEQ ID Nos: 87-124. In some embodiments, gRNAscomprising any one or more of the guide sequences of SEQ ID Nos: 5-82,or any one or more of the sgRNAs of SEQ ID Nos: 87-124, are administeredto modify the TTR gene. The guide RNAs may be administered together withan RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9) or anmRNA or vector encoding an RNA-guided DNA nuclease such as a Casnuclease (e.g., Cas9).

In some embodiments, a method of treating ATTR is provided comprisingadministering a composition comprising a guide RNA comprising any one ormore of the guide sequences of SEQ ID NOs: 5-82, or any one or more ofthe sgRNAs of SEQ ID Nos: 87-124. In some embodiments, gRNAs comprisingany one or more of the guide sequences of SEQ ID NOs: 5-82, or any oneor more of the sgRNAs of SEQ ID Nos: 87-124 are administered to treatATTR. The guide RNAs may be administered together with an RNA-guided DNAnuclease such as a Cas nuclease (e.g., Cas9) or an mRNA or vectorencoding an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9).

In some embodiments, a method of reducing TTR serum concentration isprovided comprising administering a guide RNA comprising any one or moreof the guide sequences of SEQ ID NOs: 5-82, or any one or more of thesgRNAs of SEQ ID Nos: 87-124. In some embodiments, gRNAs comprising anyone or more of the guide sequences of SEQ ID NOs: 5-82 or any one ormore of the sgRNAs of SEQ ID Nos: 87-124 are administered to reduce orprevent the accumulation of TTR in amyloids or amyloid fibrils. ThegRNAs may be administered together with an RNA-guided DNA nuclease suchas a Cas nuclease (e.g., Cas9) or an mRNA or vector encoding anRNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9).

In some embodiments, a method of reducing or preventing the accumulationof TTR in amyloids or amyloid fibrils of a subject is providedcomprising administering a composition comprising a guide RNA comprisingany one or more of the guide sequences of SEQ ID NOs: 5-82, or any oneor more of the sgRNAs of SEQ ID Nos: 87-124. In some embodiments, amethod of reducing or preventing the accumulation of TTR in amyloids oramyloid fibrils of a subject is provided comprising administering acomposition comprising any one or more of the sgRNAs of SEQ ID Nos:87-113. In some embodiments, gRNAs comprising any one or more of theguide sequences of SEQ ID NOs: 5-82 or any one or more of the sgRNAs ofSEQ ID Nos: 87-124 are administered to reduce or prevent theaccumulation of TTR in amyloids or amyloid fibrils. The gRNAs may beadministered together with an RNA-guided DNA nuclease such as a Casnuclease (e.g., Cas9) or an mRNA or vector encoding an RNA-guided DNAnuclease such as a Cas nuclease (e.g., Cas9).

In some embodiments, the gRNAs comprising the guide sequences of Table 1or one or more sgRNAs from Table 2 together with an RNA-guided DNAnuclease such as a Cas nuclease induce DSBs, and non-homologous endingjoining (NHEJ) during repair leads to a mutation in the TTR gene. Insome embodiments, NHEJ leads to a deletion or insertion of anucleotide(s), which induces a frame shift or nonsense mutation in theTTR gene.

In some embodiments, administering the guide RNAs of the invention(e.g., in a composition provided herein) reduces levels (e.g., serumlevels) of TTR in the subject, and therefore prevents accumulation andaggregation of TTR in amyloids or amyloid fibrils.

In some embodiments, reducing or preventing the accumulation of TTR inamyloids or amyloid fibrils of a subject comprises reducing orpreventing TTR deposition in one or more tissues of the subject, such asstomach, colon, or nervous tissue. In some embodiments, the nervoustissue comprises sciatic nerve or dorsal root ganglion. In someembodiments, TTR deposition is reduced in two, three, or four of thestomach, colon, dorsal root ganglion, and sciatic nerve. The level ofdeposition in a given tissue can be determined using a biopsy sample,e.g., using immunostaining. In some embodiments, reducing or preventingthe accumulation of TTR in amyloids or amyloid fibrils of a subjectand/or reducing or preventing TTR deposition is inferred based onreducing serum TTR levels for a period of time. As discussed in theexamples, it has been found that reducing serum TTR levels in accordancewith methods and uses provided herein can result in clearance ofdeposited TTR from tissues such as those discussed above and in theexamples, e.g., as measured 8 weeks after administration of thecomposition.

In some embodiments, the subject is mammalian. In some embodiments, thesubject is human. In some embodiments, the subject is cow, pig, monkey,sheep, dog, cat, fish, or poultry.

In some embodiments, the use of a guide RNAs comprising any one or moreof the guide sequences in Table 1 or one or more sgRNAs from Table 2(e.g., in a composition provided herein) is provided for the preparationof a medicament for treating a human subject having ATTR.

In some embodiments, the guide RNAs, compositions, and formulations areadministered intravenously. In some embodiments, the guide RNAs,compositions, and formulations are administered into the hepaticcirculation.

In some embodiments, a single administration of a composition comprisinga guide RNA provided herein is sufficient to knock down expression ofthe mutant protein. In some embodiments, a single administration of acomposition comprising a guide RNA provided herein is sufficient toknock out expression of the mutant protein in a population of cells. Inother embodiments, more than one administration of a compositioncomprising a guide RNA provided herein may be beneficial to maximizeediting via cumulative effects.

For example, a composition provided herein can be administered 2, 3, 4,5, or more times, such as 2 times. Administrations can be separated by aperiod of time ranging from, e.g., 1 day to 2 years, such as 1 to 7days, 7 to 14 days, 14 days to 30 days, 30 days to 60 days, 60 days to120 days, 120 days to 183 days, 183 days to 274 days, 274 days to 366days, or 366 days to 2 years.

In some embodiments, a composition is administered in an effectiveamount in the range of 0.01 to 10 mg/kg (mpk), e.g., 0.01 to 0.1 mpk,0.1 to 0.3 mpk, 0.3 to 0.5 mpk, 0.5 to 1 mpk, 1 to 2 mpk, 2 to 3 mpk, 3to 5 mpk, 5 to 10 mpk, or 0.1, 0.2, 0.3, 0.5, 1, 2, 3, 5, or 10 mpk.

In some embodiments, the efficacy of treatment with the compositions ofthe invention is seen at 1 year, 2 years, 3 years, 4 years, 5 years, or10 years after delivery. In some embodiments, efficacy of treatment withthe compositions of the invention is assessed by measuring serum levelsof TTR before and after treatment. In some embodiments, efficacy oftreatment with the compositions assessed via a reduction of serum levelsof TTR is seen at 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months,4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months,or at 11 months.

In some embodiments, treatment slows or halts disease progression.

In some embodiments, treatment slows or halts progression of FAP. Insome embodiments, treatment results in improvement, stabilization, orslowing of change in symptoms of sensorimotor neuropathy or autonomicneuropathy.

In some embodiments, treatment results in improvement, stabilization, orslowing of change in symptoms of FAC. In some embodiments, treatmentresults in improvement, stabilization, or slowing of change symptoms ofrestrictive cardiomyopathy or congestive heart failure.

In some embodiments, efficacy of treatment is measured by increasedsurvival time of the subject.

In some embodiments, efficacy of treatment is measured by improvement orslowing of progression in symptoms of sensorimotor or autonomicneuropathy. In some embodiments, efficacy of treatment is measured by anincrease or a slowing of decrease in ability to move an area of the bodyor to feel in any area of the body. In some embodiments, efficacy oftreatment is measured by improvement or a slowing of decrease in theability to swallow; breath; use arms, hands, legs, or feet; or walk. Insome embodiments, efficacy of treatment is measured by improvement or aslowing of progression of neuralgia. In some embodiments, the neuralgiais characterized by pain, burning, tingling, or abnormal feeling.

In some embodiments, efficacy of treatment is measured by improvement ora slowing of increase in postural hypotension, dizziness,gastrointestinal dysmotility, bladder dysfunction, or sexualdysfunction. In some embodiments, efficacy of treatment is measured byimprovement or a slowing of progression of weakness. In someembodiments, efficacy of treatment is measured using electromyogram,nerve conduction tests, or patient-reported outcomes.

In some embodiments, efficacy of treatment is measured by improvement orslowing of progression of symptoms of congestive heart failure or CHF.In some embodiments, efficacy of treatment is measured by an decrease ora slowing of increase in shortness of breath, trouble breathing,fatigue, or swelling in the ankles, feet, legs, abdomen, or veins in theneck. In some embodiments, efficacy of treatment is measured byimprovement or a slowing of progression of fluid buildup in the body,which may be assessed by measures such as weight gain, frequenturination, or nighttime cough. In some embodiments, efficacy oftreatment is measured using cardiac biomarker tests (such as B-typenatriuretic peptide [BNP] or N-terminal pro b-type natriuretic peptide[NT-proBNP]), lung function tests, chest x-rays, or electrocardiography.

A. Combination Therapy

In some embodiments, the invention comprises combination therapiescomprising any one of the gRNAs comprising any one or more of the guidesequences disclosed in Table 1 or any one or more of the sgRNAs in Table2 (e.g., in a composition provided herein) together with an additionaltherapy suitable for alleviating symptoms of ATTR.

In some embodiments, the additional therapy for ATTR is a treatment forsensorimotor or autonomic neuropathy. In some embodiments, the treatmentfor sensorimotor or autonomic neuropathy is a nonsteroidalanti-inflammatory drug, antidepressant, anticonvulsant medication,antiarrythmic medication, or narcotic agent. In some embodiments, theantidepressant is a tricylic agent or a serotonin-norepinephrinereuptake inhibitor. In some embodiments, the antidepressant isamitriptyline, duloxetine, or venlafaxine. In some embodiments, theanticonvulsant agent is gabapentin, pregabalin, topiramate, orcarbamazepine. In some embodiments, the additional therapy forsensorimotor neuropathy is transcutaneous electrical nerve stimulation.

In some embodiments, the additional therapy for ATTR is a treatment forrestrictive cardiomyopathy or congestive heart failure (CHF). In someembodiments, the treatment for CHF is a ACE inhibitor, aldosteroneantagonist, angiotensin receptor blocker, beta blocker, digoxin,diuretic, or isosorbide dinitrate/hydralazine hydrochloride. In someembodiments, the ACE inhibitor is enalapril, captopril, ramipril,perindopril, imidapril, or quinapril. In some embodiments, thealdosterone antagonist is eplerenone or spironolactone. In someembodiments, the angiotensin receptor blocker is azilsartan, cadesartan,eprosartan, irbesartan, losartan, olmesartan, telmisartan, or valsartan.In some embodiments, the beta blocker is acebutolol, atenolol,bisoprolol, metoprolol, nadolol, nebivolol, or propranolol. In someembodiments, the diuretic is chlorothiazide, chlorthalidone,hydrochlorothiazide, indapamide, metolazone, bumetanide, furosemide,torsemide, amiloride, or triameterene.

In some embodiments, the combination therapy comprises any one of thegRNAs comprising any one or more of the guide sequences disclosed inTable 1 or any one or more of the sgRNAs in Table 2 (e.g., in acomposition provided herein) together with a siRNA that targets TTR ormutant TTR. In some embodiments, the siRNA is any siRNA capable offurther reducing or eliminating the expression of wild type or mutantTTR. In some embodiments, the siRNA is the drug Patisiran (ALN-TTR02) orALN-TTRsc02. In some embodiments, the siRNA is administered after anyone of the gRNAs comprising any one or more of the guide sequencesdisclosed in Table 1 or any one or more of the sgRNAs in Table 2 (e.g.,in a composition provided herein). In some embodiments, the siRNA isadministered on a regular basis following treatment with any of the gRNAcompositions provided herein.

In some embodiments, the combination therapy comprises any one of thegRNAs comprising any one or more of the guide sequences disclosed inTable 1 or any one or more of the sgRNAs in Table 2 (e.g., in acomposition provided herein) together with antisense nucleotide thattargets TTR or mutant TTR. In some embodiments, the antisense nucleotideis any antisense nucleotide capable of further reducing or eliminatingthe expression of wild type or mutant TTR. In some embodiments, theantisense nucleotide is the drug Inotersen (IONS-TTRRX). In someembodiments, the antisense nucleotide is administered after any one ofthe gRNAs comprising any one or more of the guide sequences disclosed inTable 1 or any one or more of the sgRNAs in Table 2 (e.g., in acomposition provided herein). In some embodiments, the antisensenucleotide is administered on a regular basis following treatment withany of the gRNA compositions provided herein.

In some embodiments, the combination therapy comprises any one of thegRNAs comprising any one or more of the guide sequences disclosed inTable 1 or any one or more of the sgRNAs in Table 2 (e.g., in acomposition provided herein) together with a small molecule stabilizerthat promotes kinetic stabilization of the correctly folded tetramericform of TTR. In some embodiments, the small molecule stabilizer is thedrug tafamidis (Vyndaqel®) or diflunisal. In some embodiments, the smallmolecule stabilizer is administered after any one of the gRNAscomprising any one or more of the guide sequences disclosed in Table 1or any one or more of the sgRNAs in Table 2 (e.g., in a compositionprovided herein). In some embodiments, the small molecule stabilizer isadministered on a regular basis following treatment with any of the gRNAcompositions provided herein.

B. Delivery of gRNA Compositions

In some embodiments, the guide RNA compositions described herein, aloneor encoded on one or more vectors, are formulated in or administered viaa lipid nanoparticle; see e.g., PCT/US2017/024973, filed Mar. 30, 2017entitled “LIPID NANOPARTICLE FORMULATIONS FOR CRISPR/CAS COMPONENTS,”the contents of which are hereby incorporated by reference in theirentirety. Any lipid nanoparticle (LNP) known to those of skill in theart to be capable of delivering nucleotides to subjects may be utilizedwith the guide RNAs described herein, as well as either mRNA encoding anRNA-guided DNA nuclease such as Cas or Cas9, or an RNA-guided DNAnuclease such as Cas or Cas9 protein itself.

Disclosed herein are various embodiments of LNP formulations for RNAs,including CRISPR/Cas cargoes. Such LNP formulations may include (i) aCCD lipid, such as an amine lipid, (ii) a neutral lipid, (iii) a helperlipid, and (iv) a stealth lipid, such as a PEG lipid. Some embodimentsof the LNP formulations include an “amine lipid”, along with a helperlipid, a neutral lipid, and a stealth lipid such as a PEG lipid. By“lipid nanoparticle” is meant a particle that comprises a plurality of(i.e. more than one) lipid molecules physically associated with eachother by intermolecular forces.

CCD Lipids

Lipid compositions for delivery of CRISPR/Cas mRNA and guide RNAcomponents to a liver cell comprise a CCD Lipid.

In some embodiments, the CCD lipid is Lipid A, which is(9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyloctadeca-9,12-dienoate, also called3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate. Lipid A can be depicted as:

Lipid A may be synthesized according to WO2015/095340 (e.g., pp. 84-86).

In some embodiments, the CCD lipid is Lipid B, which is((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(octane-8,1-diyl)bis(decanoate),also called((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(octane-8,1-diyl)bis(decanoate). Lipid B can be depicted as:

Lipid B may be synthesized according to WO2014/136086 (e.g., pp.107-09).

In some embodiments, the CCD lipid is Lipid C, which is2-((4-(((3-(dimethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1,3-diyl(9Z,9′Z,12Z,12′Z)-bis(octadeca-9,12-dienoate). Lipid C can be depictedas:

In some embodiments, the CCD lipid is Lipid D, which is3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-(octanoyloxy)tridecyl3-octylundecanoate.

Lipid D can be depicted as:

Lipid C and Lipid D may be synthesized according to WO2015/095340.

The CCD lipid can also be an equivalent to Lipid A, Lipid B, Lipid C, orLipid D. In certain embodiments, the CCD lipid is an equivalent to LipidA, an equivalent to Lipid B, an equivalent to Lipid C, or an equivalentto Lipid D.

Amine Lipids

In some embodiments, the LNP compositions for the delivery ofbiologically active agents comprise an “amine lipid”, which is definedas Lipid A, Lipid B, Lipid C, Lipid D or equivalents of Lipid A(including acetal analogs of Lipid A), equivalents of Lipid B,equivalents of Lipid C, and equivalents of Lipid D.

In some embodiments, the amine lipid is Lipid A, which is(9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyloctadeca-9,12-dienoate, also called3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate. Lipid A can be depicted as:

Lipid A may be synthesized according to WO2015/095340 (e.g., pp. 84-86).In certain embodiments, the amine lipid is an equivalent to Lipid A.

In certain embodiments, an amine lipid is an analog of Lipid A. Incertain embodiments, a Lipid A analog is an acetal analog of Lipid A. Inparticular LNP compositions, the acetal analog is a C4-C12 acetalanalog. In some embodiments, the acetal analog is a C5-C12 acetalanalog. In additional embodiments, the acetal analog is a C5-C10 acetalanalog. In further embodiments, the acetal analog is chosen from a C4,C5, C6, C7, C9, C10, C11, and C12 acetal analog.

Amine lipids suitable for use in the LNPs described herein arebiodegradable in vivo. The amine lipids have low toxicity (e.g., aretolerated in animal models without adverse effect in amounts of greaterthan or equal to 10 mg/kg). In certain embodiments, LNPs comprising anamine lipid include those where at least 75% of the amine lipid iscleared from the plasma within 8, 10, 12, 24, or 48 hours, or 3, 4, 5,6, 7, or 10 days. In certain embodiments, LNPs comprising an amine lipidinclude those where at least 50% of the mRNA or gRNA is cleared from theplasma within 8, 10, 12, 24, or 48 hours, or 3, 4, 5, 6, 7, or 10 days.In certain embodiments, LNPs comprising an amine lipid include thosewhere at least 50% of the LNP is cleared from the plasma within 8, 10,12, 24, or 48 hours, or 3, 4, 5, 6, 7, or 10 days, for example bymeasuring a lipid (e.g. an amine lipid), RNA (e.g. mRNA), or othercomponent. In certain embodiments, lipid-encapsulated versus free lipid,RNA, or nucleic acid component of the LNP is measured.

Lipid clearance may be measured as described in literature. See Maier,M. A., et al. Biodegradable Lipids Enabling Rapidly Eliminated LipidNanoparticles for Systemic Delivery of RNAi Therapeutics. Mol. Ther.2013, 21(8), 1570-78 (“Maier”). For example, in Maier, LNP-siRNA systemscontaining luciferases-targeting siRNA were administered to six- toeight-week old male C57Bl/6 mice at 0.3 mg/kg by intravenous bolusinjection via the lateral tail vein. Blood, liver, and spleen sampleswere collected at 0.083, 0.25, 0.5, 1, 2, 4, 8, 24, 48, 96, and 168hours post-dose. Mice were perfused with saline before tissue collectionand blood samples were processed to obtain plasma. All samples wereprocessed and analyzed by LC-MS. Further, Maier describes a procedurefor assessing toxicity after administration of LNP-siRNA formulations.For example, a luciferase-targeting siRNA was administered at 0, 1, 3,5, and 10 mg/kg (5 animals/group) via single intravenous bolus injectionat a dose volume of 5 mL/kg to male Sprague-Dawley rats. After 24 hours,about 1 mL of blood was obtained from the jugular vein of consciousanimals and the serum was isolated. At 72 hours post-dose, all animalswere euthanized for necropsy. Assessment of clinical signs, body weight,serum chemistry, organ weights and histopathology was performed.Although Maier describes methods for assessing siRNA-LNP formulations,these methods may be applied to assess clearance, pharmacokinetics, andtoxicity of administration of LNP compositions of the presentdisclosure.

The amine lipids lead to an increased clearance rate. In someembodiments, the clearance rate is a lipid clearance rate, for examplethe rate at which an amine lipid is cleared from the blood, serum, orplasma. In some embodiments, the clearance rate is an RNA clearancerate, for example the rate at which an mRNA or a gRNA is cleared fromthe blood, serum, or plasma. In some embodiments, the clearance rate isthe rate at which LNP is cleared from the blood, serum, or plasma. Insome embodiments, the clearance rate is the rate at which LNP is clearedfrom a tissue, such as liver tissue or spleen tissue. In certainembodiments, a high rate of clearance rate leads to a safety profilewith no substantial adverse effects. The amine lipids reduce LNPaccumulation in circulation and in tissues. In some embodiments, areduction in LNP accumulation in circulation and in tissues leads to asafety profile with no substantial adverse effects.

The amine lipids of the present disclosure may be ionizable dependingupon the pH of the medium they are in. For example, in a slightly acidicmedium, the amine lipids may be protonated and thus bear a positivecharge. Conversely, in a slightly basic medium, such as, for example,blood where pH is approximately 7.35, the amine lipids may not beprotonated and thus bear no charge. In some embodiments, the aminelipids of the present disclosure may be protonated at a pH of at leastabout 9. In some embodiments, the amine lipids of the present disclosuremay be protonated at a pH of at least about 9. In some embodiments, theamine lipids of the present disclosure may be protonated at a pH of atleast about 10.

The ability of an amine lipid to bear a charge is related to itsintrinsic pKa. For example, the amine lipids of the present disclosuremay each, independently, have a pKa in the range of from about 5.8 toabout 6.2. For example, the amine lipids of the present disclosure mayeach, independently, have a pKa in the range of from about 5.8 to about6.5. This may be advantageous as it has been found that cationic lipidswith a pKa ranging from about 5.1 to about 7.4 are effective fordelivery of cargo in vivo, e.g. to the liver. Further, it has been foundthat cationic lipids with a pKa ranging from about 5.3 to about 6.4 areeffective for delivery in vivo, e.g. to tumors. See, e.g.,WO2014/136086.

Additional Lipids

“Neutral lipids” suitable for use in a lipid composition of thedisclosure include, for example, a variety of neutral, uncharged orzwitterionic lipids. Examples of neutral phospholipids suitable for usein the present disclosure include, but are not limited to,5-heptadecylbenzene-1,3-diol (resorcinol),dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine(DSPC), pohsphocholine (DOPC), dimyristoylphosphatidylcholine (DMPC),phosphatidylcholine (PLPC), 1,2-distearoyl-sn-glycero-3-phosphocholine(DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC),dilauryloylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine(DMPC), 1-myristoyl-2-palmitoyl phosphatidylcholine (MPPC),1-palmitoyl-2-myristoyl phosphatidylcholine (PMPC),1-palmitoyl-2-stearoyl phosphatidylcholine (PSPC),1,2-diarachidoyl-sn-glycero-3-phosphocholine (DBPC),1-stearoyl-2-palmitoyl phosphatidylcholine (SPPC),1,2-dieicosenoyl-sn-glycero-3-phosphocholine (DEPC), palmitoyloleoylphosphatidylcholine (POPC), lysophosphatidyl choline, dioleoylphosphatidylethanolamine (DOPE), dilinoleoylphosphatidylcholine distearoylphosphatidylethanolamine (DSPE), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine(DPPE), palmitoyloleoyl phosphatidylethanolamine (POPE),lysophosphatidylethanolamine and combinations thereof. In oneembodiment, the neutral phospholipid may be selected from the groupconsisting of distearoylphosphatidylcholine (DSPC) and dimyristoylphosphatidyl ethanolamine (DMPE). In another embodiment, the neutralphospholipid may be distearoylphosphatidylcholine (DSPC).

“Helper lipids” include steroids, sterols, and alkyl resorcinols. Helperlipids suitable for use in the present disclosure include, but are notlimited to, cholesterol, 5-heptadecylresorcinol, and cholesterolhemisuccinate. In one embodiment, the helper lipid may be cholesterol.In one embodiment, the helper lipid may be cholesterol hemisuccinate.

“Stealth lipids” are lipids that alter the length of time thenanoparticles can exist in vivo (e.g., in the blood). Stealth lipids mayassist in the formulation process by, for example, reducing particleaggregation and controlling particle size. Stealth lipids used hereinmay modulate pharmacokinetic properties of the LNP. Stealth lipidssuitable for use in a lipid composition of the disclosure include, butare not limited to, stealth lipids having a hydrophilic head grouplinked to a lipid moiety. Stealth lipids suitable for use in a lipidcomposition of the present disclosure and information about thebiochemistry of such lipids can be found in Romberg et al.,Pharmaceutical Research, Vol. 25, No. 1, 2008, pg. 55-71 and Hoekstra etal., Biochimica et Biophysica Acta 1660 (2004) 41-52. Additionalsuitable PEG lipids are disclosed, e.g., in WO 2006/007712.

In one embodiment, the hydrophilic head group of stealth lipid comprisesa polymer moiety selected from polymers based on PEG. Stealth lipids maycomprise a lipid moiety. In some embodiments, the stealth lipid is a PEGlipid.

In one embodiment, a stealth lipid comprises a polymer moiety selectedfrom polymers based on PEG (sometimes referred to as poly(ethyleneoxide)), poly(oxazoline), poly(vinyl alcohol), poly(glycerol),poly(N-vinylpyrrolidone), polyaminoacids andpoly[N-(2-hydroxypropyl)methacrylamide].

In one embodiment, the PEG lipid comprises a polymer moiety based on PEG(sometimes referred to as poly(ethylene oxide)).

The PEG lipid further comprises a lipid moiety. In some embodiments, thelipid moiety may be derived from diacylglycerol or diacylglycamide,including those comprising a dialkylglycerol or dialkylglycamide grouphaving alkyl chain length independently comprising from about C4 toabout C40 saturated or unsaturated carbon atoms, wherein the chain maycomprise one or more functional groups such as, for example, an amide orester. In some embodiments, the alkyl chail length comprises about C10to C20. The dialkylglycerol or dialkylglycamide group can furthercomprise one or more substituted alkyl groups. The chain lengths may besymmetrical or assymetric.

Unless otherwise indicated, the term “PEG” as used herein means anypolyethylene glycol or other polyalkylene ether polymer. In oneembodiment, PEG is an optionally substituted linear or branched polymerof ethylene glycol or ethylene oxide. In one embodiment, PEG isunsubstituted. In one embodiment, the PEG is substituted, e.g., by oneor more alkyl, alkoxy, acyl, hydroxy, or aryl groups. In one embodiment,the term includes PEG copolymers such as PEG-polyurethane orPEG-polypropylene (see, e.g., J. Milton Harris, Poly(ethylene glycol)chemistry: biotechnical and biomedical applications (1992)); in anotherembodiment, the term does not include PEG copolymers. In one embodiment,the PEG has a molecular weight of from about 130 to about 50,000, in asub-embodiment, about 150 to about 30,000, in a sub-embodiment, about150 to about 20,000, in a sub-embodiment about 150 to about 15,000, in asub-embodiment, about 150 to about 10,000, in a sub-embodiment, about150 to about 6,000, in a sub-embodiment, about 150 to about 5,000, in asub-embodiment, about 150 to about 4,000, in a sub-embodiment, about 150to about 3,000, in a sub-embodiment, about 300 to about 3,000, in asub-embodiment, about 1,000 to about 3,000, and in a sub-embodiment,about 1,500 to about 2,500.

In certain embodiments, the PEG (e.g., conjugated to a lipid moiety orlipid, such as a stealth lipid), is a “PEG-2K,” also termed “PEG 2000,”which has an average molecular weight of about 2,000 daltons. PEG-2K isrepresented herein by the following formula (I), wherein n is 45,meaning that the number averaged degree of polymerization comprisesabout 45 subunits. However, other PEG embodiments known in the art maybe used, including, e.g., those where the number-averaged degree ofpolymerization comprises about 23 subunits (n=23), and/or 68 subunits(n=68). In some embodiments, n may range from about 30 to about 60. Insome embodiments, n may range from about 35 to about 55. In someembodiments, n may range from about 40 to about 50. In some embodiments,n may range from about 42 to about 48. In some embodiments, n may be 45.In some embodiments, R may be selected from H, substituted alkyl, andunsubstituted alkyl. In some embodiments, R may be unsubstituted alkyl.In some embodiments, R may be methyl.

In any of the embodiments described herein, the PEG lipid may beselected from PEG-dilauroylglycerol, PEG-dimyristoylglycerol (PEG-DMG)(catalog # GM-020 from NOF, Tokyo, Japan), PEG-dipalmitoylglycerol,PEG-di stearoylglycerol (PEG-DSPE) (catalog # DSPE-020CN, NOF, Tokyo,Japan), PEG-dilaurylglycamide, PEG-dimyristylglycamide,PEG-dipalmitoylglycamide, and PEG-di stearoylglycamide, PEG-cholesterol(1-[8′-(Cholest-5-en-3[beta]-oxy)carboxamido-3′,6′-dioxaoctanyl]carbamoyl-[omega]-methyl-poly(ethyleneglycol), PEG-DMB (3,4-ditetradecoxylbenzyl-[omega]-methyl-poly(ethyleneglycol)ether),1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (PEG2k-DMG) (cat. #880150P from Avanti Polar Lipids,Alabaster, Ala., USA),1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (PEG2k-DSPE) (cat. #880120C from Avanti Polar Lipids,Alabaster, Ala., USA), 1,2-distearoyl-sn-glycerol, methoxypolyethyleneglycol (PEG2k-DSG; GS-020, NOF Tokyo, Japan), poly(ethyleneglycol)-2000-dimethacrylate (PEG2k-DMA), and1,2-distearyloxypropyl-3-amine-N-[methoxy(polyethylene glycol)-2000](PEG2k-DSA). In one embodiment, the PEG lipid may be PEG2k-DMG. In someembodiments, the PEG lipid may be PEG2k-DSG. In one embodiment, the PEGlipid may be PEG2k-DSPE. In one embodiment, the PEG lipid may bePEG2k-DMA. In one embodiment, the PEG lipid may be PEG2k-C-DMA. In oneembodiment, the PEG lipid may be compound 5027, disclosed inWO2016/010840 (paragraphs [00240] to [00244]). In one embodiment, thePEG lipid may be PEG2k-DSA. In one embodiment, the PEG lipid may bePEG2k-C11. In some embodiments, the PEG lipid may be PEG2k-C14. In someembodiments, the PEG lipid may be PEG2k-C16. In some embodiments, thePEG lipid may be PEG2k-C18.

LNP Formulations

The LNP may contain (i) an amine lipid for encapsulation and forendosomal escape, (ii) a neutral lipid for stabilization, (iii) a helperlipid, also for stabilization, and (iv) a stealth lipid, such as a PEGlipid.

In some embodiments, an LNP composition may comprise an RNA componentthat includes one or more of an RNA-guided DNA-binding agent, a Casnuclease mRNA, a Class 2 Cas nuclease mRNA, a Cas9 mRNA, and a gRNA. Insome embodiments, an LNP composition may include a Class 2 Cas nucleaseand a gRNA as the RNA component. In certain embodiments, an LNPcomposition may comprise the RNA component, an amine lipid, a helperlipid, a neutral lipid, and a stealth lipid. In certain LNPcompositions, the helper lipid is cholesterol. In other compositions,the neutral lipid is DSPC. In additional embodiments, the stealth lipidis PEG2k-DMG or PEG2k-C11. In certain embodiments, the LNP compositioncomprises Lipid A or an equivalent of Lipid A; a helper lipid; a neutrallipid; a stealth lipid; and a guide RNA. In certain compositions, theamine lipid is Lipid A. In certain compositions, the amine lipid isLipid A or an acetal analog thereof; the helper lipid is cholesterol;the neutral lipid is DSPC; and the stealth lipid is PEG2k-DMG.

In certain embodiments, lipid compositions are described according tothe respective molar ratios of the component lipids in the formulation.Embodiments of the present disclosure provide lipid compositionsdescribed according to the respective molar ratios of the componentlipids in the formulation. In one embodiment, the mol-% of the aminelipid may be from about 30 mol-% to about 60 mol-%. In one embodiment,the mol-% of the amine lipid may be from about 40 mol-% to about 60mol-%. In one embodiment, the mol-% of the amine lipid may be from about45 mol-% to about 60 mol-%. In one embodiment, the mol-% of the aminelipid may be from about 50 mol-% to about 60 mol-%. In one embodiment,the mol-% of the amine lipid may be from about 55 mol-% to about 60mol-%. In one embodiment, the mol-% of the amine lipid may be from about50 mol-% to about 55 mol-%. In one embodiment, the mol-% of the aminelipid may be about 50 mol-%. In one embodiment, the mol-% of the aminelipid may be about 55 mol-%. In some embodiments, the amine lipid mol-%of the LNP batch will be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% ofthe target mol-%. In some embodiments, the amine lipid mol-% of the LNPbatch will be ±4 mol-%, ±3 mol-%, ±2 mol-%, ±1.5 mol-%, ±1 mol-%, ±0.5mol-%, or ±0.25 mol-% of the target mol-%. All mol-% numbers are givenas a fraction of the lipid component of the LNP compositions. In certainembodiments, LNP inter-lot variability of the amine lipid mol-% will beless than 15%, less than 10% or less than 5%.

In one embodiment, the mol-% of the neutral lipid may be from about 5mol-% to about 15 mol-%. In one embodiment, the mol-% of the neutrallipid may be from about 7 mol-% to about 12 mol-%. In one embodiment,the mol-% of the neutral lipid may be about 9 mol-%. In someembodiments, the neutral lipid mol-% of the LNP batch will be ±30%,±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the target neutral lipid mol-%.In certain embodiments, LNP inter-lot variability will be less than 15%,less than 10% or less than 5%.

In one embodiment, the mol-% of the helper lipid may be from about 20mol-% to about 60 mol-%. In one embodiment, the mol-% of the helperlipid may be from about 25 mol-% to about 55 mol-%. In one embodiment,the mol-% of the helper lipid may be from about 25 mol-% to about 50mol-%. In one embodiment, the mol-% of the helper lipid may be fromabout 25 mol-% to about 40 mol-%. In one embodiment, the mol-% of thehelper lipid may be from about 30 mol-% to about 50 mol-%. In oneembodiment, the mol-% of the helper lipid may be from about 30 mol-% toabout 40 mol-%. In one embodiment, the mol-% of the helper lipid isadjusted based on amine lipid, neutral lipid, and PEG lipidconcentrations to bring the lipid component to 100 mol-%. In someembodiments, the helper mol-% of the LNP batch will be ±30%, ±25%, ±20%,±15%, ±10%, ±5%, or ±2.5% of the target mol-%. In certain embodiments,LNP inter-lot variability will be less than 15%, less than 10% or lessthan 5%.

In one embodiment, the mol-% of the PEG lipid may be from about 1 mol-%to about 10 mol-%. In one embodiment, the mol-% of the PEG lipid may befrom about 2 mol-% to about 10 mol-%. In one embodiment, the mol-% ofthe PEG lipid may be from about 2 mol-% to about 8 mol-%. In oneembodiment, the mol-% of the PEG lipid may be from about 2 mol-% toabout 4 mol-%. In one embodiment, the mol-% of the PEG lipid may be fromabout 2.5 mol-% to about 4 mol-%. In one embodiment, the mol-% of thePEG lipid may be about 3 mol-%. In one embodiment, the mol-% of the PEGlipid may be about 2.5 mol-%. In some embodiments, the PEG lipid mol-%of the LNP batch will be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% ofthe target PEG lipid mol-%. In certain embodiments, LNP inter-lotvariability will be less than 15%, less than 10% or less than 5%.

In certain embodiments, the cargo includes an mRNA encoding anRNA-guided DNA-binding agent (e.g. a Cas nuclease, a Class 2 Casnuclease, or Cas9), and a gRNA or a nucleic acid encoding a gRNA, or acombination of mRNA and gRNA. In one embodiment, an LNP composition maycomprise a Lipid A or its equivalents. In some aspects, the amine lipidis Lipid A. In some aspects, the amine lipid is a Lipid A equivalent,e.g. an analog of Lipid A. In certain aspects, the amine lipid is anacetal analog of Lipid A. In various embodiments, an LNP compositioncomprises an amine lipid, a neutral lipid, a helper lipid, and a PEGlipid. In certain embodiments, the helper lipid is cholesterol. Incertain embodiments, the neutral lipid is DSPC. In specific embodiments,PEG lipid is PEG2k-DMG. In some embodiments, an LNP composition maycomprise a Lipid A, a helper lipid, a neutral lipid, and a PEG lipid. Insome embodiments, an LNP composition comprises an amine lipid, DSPC,cholesterol, and a PEG lipid. In some embodiments, the LNP compositioncomprises a PEG lipid comprising DMG. In certain embodiments, the aminelipid is selected from Lipid A, and an equivalent of Lipid A, includingan acetal analog of Lipid A. In additional embodiments, an LNPcomposition comprises Lipid A, cholesterol, DSPC, and PEG2k-DMG.

Embodiments of the present disclosure also provide lipid compositionsdescribed according to the molar ratio between the positively chargedamine groups of the amine lipid (N) and the negatively charged phosphategroups (P) of the nucleic acid to be encapsulated. This may bemathematically represented by the equation N/P. In some embodiments, anLNP composition may comprise a lipid component that comprises an aminelipid, a helper lipid, a neutral lipid, and a helper lipid; and anucleic acid component, wherein the N/P ratio is about 3 to 10. In someembodiments, an LNP composition may comprise a lipid component thatcomprises an amine lipid, a helper lipid, a neutral lipid, and a helperlipid; and an RNA component, wherein the N/P ratio is about 3 to 10. Inone embodiment, the N/P ratio may about 5-7. In one embodiment, the N/Pratio may about 4.5-8. In one embodiment, the N/P ratio may about 6. Inone embodiment, the N/P ratio may be 6±1. In one embodiment, the N/Pratio may about 6±0.5. In some embodiments, the N/P ratio will be ±30%,±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the target N/P ratio. Incertain embodiments, LNP inter-lot variability will be less than 15%,less than 10% or less than 5%.

In some embodiments, the RNA component may comprise an mRNA, such as anmRNA disclosed herein, e.g., encoding a Cas nuclease. In one embodiment,RNA component may comprise a Cas9 mRNA. In some compositions comprisingan mRNA encoding a Cas nuclease, the LNP further comprises a gRNAnucleic acid, such as a gRNA. In some embodiments, the RNA componentcomprises a Cas nuclease mRNA and a gRNA. In some embodiments, the RNAcomponent comprises a Class 2 Cas nuclease mRNA and a gRNA.

In certain embodiments, an LNP composition may comprise an mRNAdisclosed herein, e.g., encoding a Cas nuclease, such as a Class 2 Casnuclease, an amine lipid, a helper lipid, a neutral lipid, and a PEGlipid. In certain LNP compositions comprising an mRNA encoding a Casnuclease such as a Class 2 Cas nuclease, the helper lipid ischolesterol. In other compositions comprising an mRNA encoding a Casnuclease such as a Class 2 Cas nuclease, the neutral lipid is DSPC. Inadditional embodiments comprising an mRNA encoding a Cas nuclease suchas a Class 2 Cas nuclease, the PEG lipid is PEG2k-DMG or PEG2k-C11. Inspecific compositions comprising an mRNA encoding a Cas nuclease such asa Class 2 Cas nuclease, the amine lipid is selected from Lipid A and itsequivalents, such as an acetal analog of Lipid A.

In some embodiments, an LNP composition may comprise a gRNA. In certainembodiments, an LNP composition may comprise an amine lipid, a gRNA, ahelper lipid, a neutral lipid, and a PEG lipid. In certain LNPcompositions comprising a gRNA, the helper lipid is cholesterol. In somecompositions comprising a gRNA, the neutral lipid is DSPC. In additionalembodiments comprising a gRNA, the PEG lipid is PEG2k-DMG or PEG2k-C11.In certain embodiments, the amine lipid is selected from Lipid A and itsequivalents, such as an acetal analog of Lipid A.

In one embodiment, an LNP composition may comprise an sgRNA. In oneembodiment, an LNP composition may comprise a Cas9 sgRNA. In oneembodiment, an LNP composition may comprise a Cpf1 sgRNA. In somecompositions comprising an sgRNA, the LNP includes an amine lipid, ahelper lipid, a neutral lipid, and a PEG lipid. In certain compositionscomprising an sgRNA, the helper lipid is cholesterol. In othercompositions comprising an sgRNA, the neutral lipid is DSPC. Inadditional embodiments comprising an sgRNA, the PEG lipid is PEG2k-DMGor PEG2k-C11. In certain embodiments, the amine lipid is selected fromLipid A and its equivalents, such as acetal analogs of Lipid A.

In certain embodiments, an LNP composition comprises an mRNA encoding aCas nuclease and a gRNA, which may be an sgRNA. In one embodiment, anLNP composition may comprise an amine lipid, an mRNA encoding a Casnuclease, a gRNA, a helper lipid, a neutral lipid, and a PEG lipid. Incertain compositions comprising an mRNA encoding a Cas nuclease and agRNA, the helper lipid is cholesterol. In some compositions comprisingan mRNA encoding a Cas nuclease and a gRNA, the neutral lipid is DSPC.In additional embodiments comprising an mRNA encoding a Cas nuclease anda gRNA, the PEG lipid is PEG2k-DMG or PEG2k-C11. In certain embodiments,the amine lipid is selected from Lipid A and its equivalents, such asacetal analogs of Lipid A.

In certain embodiments, the LNP compositions include a Cas nucleasemRNA, such as a Class 2 Cas mRNA and at least one gRNA. In certainembodiments, the LNP composition includes a ratio of gRNA to Casnuclease mRNA, such as Class 2 Cas nuclease mRNA from about 25:1 toabout 1:25. In certain embodiments, the LNP formulation includes a ratioof gRNA to Cas nuclease mRNA, such as Class 2 Cas nuclease mRNA fromabout 10:1 to about 1:10. In certain embodiments, the LNP formulationincludes a ratio of gRNA to Cas nuclease mRNA, such as Class 2 Casnuclease mRNA from about 8:1 to about 1:8. As measured herein, theratios are by weight. In some embodiments, the LNP formulation includesa ratio of gRNA to Cas nuclease mRNA, such as Class 2 Cas mRNA fromabout 5:1 to about 1:5. In some embodiments, ratio range is about 3:1 to1:3, about 2:1 to 1:2, about 5:1 to 1:2, about 5:1 to 1:1, about 3:1 to1:2, about 3:1 to 1:1, about 3:1, about 2:1 to 1:1. In some embodiments,the gRNA to mRNA ratio is about 3:1 or about 2:1 In some embodiments theratio of gRNA to Cas nuclease mRNA, such as Class 2 Cas nuclease isabout 1:1. The ratio may be about 25:1, 10:1, 5:1, 3:1, 1:1, 1:3, 1:5,1:10, or 1:25.

The LNP compositions disclosed herein may include a template nucleicacid. The template nucleic acid may be co-formulated with an mRNAencoding a Cas nuclease, such as a Class 2 Cas nuclease mRNA. In someembodiments, the template nucleic acid may be co-formulated with a guideRNA. In some embodiments, the template nucleic acid may be co-formulatedwith both an mRNA encoding a Cas nuclease and a guide RNA. In someembodiments, the template nucleic acid may be formulated separately froman mRNA encoding a Cas nuclease or a guide RNA. The template nucleicacid may be delivered with, or separately from the LNP compositions. Insome embodiments, the template nucleic acid may be single- ordouble-stranded, depending on the desired repair mechanism. The templatemay have regions of homology to the target DNA, or to sequences adjacentto the target DNA.

In some embodiments, LNPs are formed by mixing an aqueous RNA solutionwith an organic solvent-based lipid solution, e.g., 100% ethanol.Suitable solutions or solvents include or may contain: water, PBS, Trisbuffer, NaCl, citrate buffer, ethanol, chloroform, diethylether,cyclohexane, tetrahydrofuran, methanol, isopropanol. A pharmaceuticallyacceptable buffer, e.g., for in vivo administration of LNPs, may beused. In certain embodiments, a buffer is used to maintain the pH of thecomposition comprising LNPs at or above pH 6.5. In certain embodiments,a buffer is used to maintain the pH of the composition comprising LNPsat or above pH 7.0. In certain embodiments, the composition has a pHranging from about 7.2 to about 7.7. In additional embodiments, thecomposition has a pH ranging from about 7.3 to about 7.7 or ranging fromabout 7.4 to about 7.6. In further embodiments, the composition has a pHof about 7.2, 7.3, 7.4, 7.5, 7.6, or 7.7. The pH of a composition may bemeasured with a micro pH probe. In certain embodiments, a cryoprotectantis included in the composition. Non-limiting examples of cryoprotectantsinclude sucrose, trehalose, glycerol, DMSO, and ethylene glycol.Exemplary compositions may include up to 10% cryoprotectant, such as,for example, sucrose. In certain embodiments, the LNP composition mayinclude about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% cryoprotectant. Incertain embodiments, the LNP composition may include about 1, 2, 3, 4,5, 6, 7, 8, 9, or 10% sucrose. In some embodiments, the LNP compositionmay include a buffer. In some embodiments, the buffer may comprise aphosphate buffer (PBS), a Tris buffer, a citrate buffer, and mixturesthereof. In certain exemplary embodiments, the buffer comprises NaCl. Incertain embodiments, NaCl is omitted. Exemplary amounts of NaCl mayrange from about 20 mM to about 45 mM. Exemplary amounts of NaCl mayrange from about 40 mM to about 50 mM. In some embodiments, the amountof NaCl is about 45 mM. In some embodiments, the buffer is a Trisbuffer. Exemplary amounts of Tris may range from about 20 mM to about 60mM. Exemplary amounts of Tris may range from about 40 mM to about 60 mM.In some embodiments, the amount of Tris is about 50 mM. In someembodiments, the buffer comprises NaCl and Tris. Certain exemplaryembodiments of the LNP compositions contain 5% sucrose and 45 mM NaCl inTris buffer. In other exemplary embodiments, compositions containsucrose in an amount of about 5% w/v, about 45 mM NaCl, and about 50 mMTris at pH 7.5. The salt, buffer, and cryoprotectant amounts may bevaried such that the osmolality of the overall formulation ismaintained. For example, the final osmolality may be maintained at lessthan 450 mOsm/L. In further embodiments, the osmolality is between 350and 250 mOsm/L. Certain embodiments have a final osmolality of 300+/−20mOsm/L.

In some embodiments, microfluidic mixing, T-mixing, or cross-mixing isused. In certain aspects, flow rates, junction size, junction geometry,junction shape, tube diameter, solutions, and/or RNA and lipidconcentrations may be varied. LNPs or LNP compositions may beconcentrated or purified, e.g., via dialysis, tangential flowfiltration, or chromatography. The LNPs may be stored as a suspension,an emulsion, or a lyophilized powder, for example. In some embodiments,an LNP composition is stored at 2-8° C., in certain aspects, the LNPcompositions are stored at room temperature. In additional embodiments,an LNP composition is stored frozen, for example at −20° C. or −80° C.In other embodiments, an LNP composition is stored at a temperatureranging from about 0° C. to about −80° C. Frozen LNP compositions may bethawed before use, for example on ice, at 4° C., at room temperature, orat 25° C. Frozen LNP compositions may be maintained at varioustemperatures, for example on ice, at 4° C., at room temperature, at 25°C., or at 37° C.

In some embodiments, an LNP composition has greater than about 80%encapsulation. In some embodiments, an LNP composition has a particlesize less than about 120 nm. In some embodiments, an LNP composition hasa pdi less than about 0.2. In some embodiments, at least two of thesefeatures are present. In some embodiments, each of these three featuresis present. Analytical methods for determining these parameters arediscussed below in the general reagents and methods section.

In some embodiments, microfluidic mixing, T-mixing, or cross-mixing isused. In certain aspects, flow rates, junction size, junction geometry,junction shape, tube diameter, solutions, and/or RNA and lipidconcentrations may be varied. LNPs or LNP compositions may beconcentrated or purified, e.g., via dialysis or chromatography. The LNPsmay be stored as a suspension, an emulsion, or a lyophilized powder, forexample. In some embodiments, the LNP compositions are stored at 2-8°C., in certain aspects, the LNP compositions are stored at roomtemperature. In additional embodiments, the LNP composition is storedfrozen, for example at −20° C. or −80° C. In other embodiments, the LNPcomposition is stored at a temperature ranging from 0° C. to −80° C.Frozen LNP compositions may be thawed before use, for example on ice, atroom temperature, or at 25° C.

Dynamic Light Scattering (“DLS”) can be used to characterize thepolydispersity index (“pdi”) and size of the LNPs of the presentdisclosure. DLS measures the scattering of light that results fromsubjecting a sample to a light source. PDI, as determined from DLSmeasurements, represents the distribution of particle size (around themean particle size) in a population, with a perfectly uniform populationhaving a PDI of zero. In some embodiments, the pdi may range from 0.005to 0.75. In some embodiments, the pdi may range from 0.01 to 0.5. Insome embodiments, the pdi may range from 0.02 to 0.4. In someembodiments, the pdi may range from 0.03 to 0.35. In some embodiments,the pdi may range from 0.1 to 0.35.

In some embodiments, LNPs disclosed herein have a size of 1 to 250 nm.In some embodiments, the LNPs have a size of 10 to 200 nm. In furtherembodiments, the LNPs have a size of 20 to 150 nm. In some embodiments,the LNPs have a size of 50 to 150 nm. In some embodiments, the LNPs havea size of 50 to 100 nm. In some embodiments, the LNPs have a size of 50to 120 nm. In some embodiments, the LNPs have a size of 75 to 150 nm. Insome embodiments, the LNPs have a size of 30 to 200 nm. Unless indicatedotherwise, all sizes referred to herein are the average sizes(diameters) of the fully formed nanoparticles, as measured by dynamiclight scattering on a Malvern Zetasizer. The nanoparticle sample isdiluted in phosphate buffered saline (PBS) so that the count rate isapproximately 200-400 kcts. The data is presented as a weighted-averageof the intensity measure. In some embodiments, the LNPs are formed withan average encapsulation efficiency ranging from 50% to 100%. In someembodiments, the LNPs are formed with an average encapsulationefficiency ranging from 50% to 70%. In some embodiments, the LNPs areformed with an average encapsulation efficiency ranging from 70% to 90%.In some embodiments, the LNPs are formed with an average encapsulationefficiency ranging from 90% to 100%. In some embodiments, the LNPs areformed with an average encapsulation efficiency ranging from 75% to 95%.

In some embodiments, LNPs associated with the gRNAs disclosed herein arefor use in preparing a medicament for treating ATTR. In someembodiments, LNPs associated with the gRNAs disclosed herein are for usein preparing a medicament for reducing or preventing accumulation andaggregation of TTR in amyloids or amyloid fibrils in subjects havingATTR. In some embodiments, LNPs associated with the gRNAs disclosedherein are for use in preparing a medicament for reducing serum TTRconcentration. In some embodiments, LNPs associated with the gRNAsdisclosed herein are for use in treating ATTR in a subject, such as amammal, e.g., a primate such as a human. In some embodiments, LNPsassociated with the gRNAs disclosed herein are for use in reducing orpreventing accumulation and aggregation of TTR in amyloids or amyloidfibrils in subjects having ATTR, such as a mammal, e.g., a primate suchas a human. In some embodiments, LNPs associated with the gRNAsdisclosed herein are for use in reducing serum TTR concentration in asubject, such as a mammal, e.g., a primate such as a human.

Electroporation is also a well-known means for delivery of cargo, andany electroporation methodology may be used for delivery of any one ofthe gRNAs disclosed herein. In some embodiments, electroporation may beused to deliver any one of the gRNAs disclosed herein and an RNA-guidedDNA nuclease such as Cas9 or an mRNA encoding an RNA-guided DNA nucleasesuch as Cas9.

In some embodiments, the invention comprises a method for delivering anyone of the gRNAs disclosed herein to an ex vivo cell, wherein the gRNAis associated with an LNP or not associated with an LNP. In someembodiments, the gRNA/LNP or gRNA is also associated with an RNA-guidedDNA nuclease such as Cas9 or an mRNA encoding an RNA-guided DNA nucleasesuch as Cas9.

In certain embodiments, the invention comprises DNA or RNA vectorsencoding any of the guide RNAs comprising any one or more of the guidesequences described herein. In some embodiments, in addition to guideRNA sequences, the vectors further comprise nucleic acids that do notencode guide RNAs. Nucleic acids that do not encode guide RNA include,but are not limited to, promoters, enhancers, regulatory sequences, andnucleic acids encoding an RNA-guided DNA nuclease, which can be anuclease such as Cas9. In some embodiments, the vector comprises one ormore nucleotide sequence(s) encoding a crRNA, a trRNA, or a crRNA andtrRNA. In some embodiments, the vector comprises one or more nucleotidesequence(s) encoding a sgRNA and an mRNA encoding an RNA-guided DNAnuclease, which can be a Cas nuclease, such as Cas9 or Cpf1. In someembodiments, the vector comprises one or more nucleotide sequence(s)encoding a crRNA, a trRNA, and an mRNA encoding an RNA-guided DNAnuclease, which can be a Cas protein, such as, Cas9. In one embodiment,the Cas9 is from Streptococcus pyogenes (i.e., Spy Cas9). In someembodiments, the nucleotide sequence encoding the crRNA, trRNA, or crRNAand trRNA (which may be a sgRNA) comprises or consists of a guidesequence flanked by all or a portion of a repeat sequence from anaturally-occurring CRISPR/Cas system. The nucleic acid comprising orconsisting of the crRNA, trRNA, or crRNA and trRNA may further comprisea vector sequence wherein the vector sequence comprises or consists ofnucleic acids that are not naturally found together with the crRNA,trRNA, or crRNA and trRNA.

In some embodiments, the crRNA and the trRNA are encoded bynon-contiguous nucleic acids within one vector. In other embodiments,the crRNA and the trRNA may be encoded by a contiguous nucleic acid. Insome embodiments, the crRNA and the trRNA are encoded by oppositestrands of a single nucleic acid. In other embodiments, the crRNA andthe trRNA are encoded by the same strand of a single nucleic acid.

In some embodiments, the vector may be circular. In other embodiments,the vector may be linear. In some embodiments, the vector may beenclosed in a lipid nanoparticle, liposome, non-lipid nanoparticle, orviral capsid. Non-limiting exemplary vectors include plasmids,phagemids, cosmids, artificial chromosomes, minichromosomes,transposons, viral vectors, and expression vectors.

In some embodiments, the vector may be a viral vector. In someembodiments, the viral vector may be genetically modified from its wildtype counterpart. For example, the viral vector may comprise aninsertion, deletion, or substitution of one or more nucleotides tofacilitate cloning or such that one or more properties of the vector ischanged. Such properties may include packaging capacity, transductionefficiency, immunogenicity, genome integration, replication,transcription, and translation. In some embodiments, a portion of theviral genome may be deleted such that the virus is capable of packagingexogenous sequences having a larger size. In some embodiments, the viralvector may have an enhanced transduction efficiency. In someembodiments, the immune response induced by the virus in a host may bereduced. In some embodiments, viral genes (such as, e.g., integrase)that promote integration of the viral sequence into a host genome may bemutated such that the virus becomes non-integrating. In someembodiments, the viral vector may be replication defective. In someembodiments, the viral vector may comprise exogenous transcriptional ortranslational control sequences to drive expression of coding sequenceson the vector. In some embodiments, the virus may be helper-dependent.For example, the virus may need one or more helper virus to supply viralcomponents (such as, e.g., viral proteins) required to amplify andpackage the vectors into viral particles. In such a case, one or morehelper components, including one or more vectors encoding the viralcomponents, may be introduced into a host cell along with the vectorsystem described herein. In other embodiments, the virus may behelper-free. For example, the virus may be capable of amplifying andpackaging the vectors without any helper virus. In some embodiments, thevector system described herein may also encode the viral componentsrequired for virus amplification and packaging.

Non-limiting exemplary viral vectors include adeno-associated virus(AAV) vector, lentivirus vectors, adenovirus vectors, helper dependentadenoviral vectors (HDAd), herpes simplex virus (HSV-1) vectors,bacteriophage T4, baculovirus vectors, and retrovirus vectors. In someembodiments, the viral vector may be an AAV vector. In some embodiments,the viral vector is AAV2, AAV3, AAV3B, AAV5, AAV6, AAV6.2, AAV7,AAVrh.64R1, AAVhu.37, AAVrh.8, AAVrh.32.33, AAV8, AAV9, AAVrh10, orAAVLK03. In other embodiments, the viral vector may a lentivirus vector.

In some embodiments, the lentivirus may be non-integrating. In someembodiments, the viral vector may be an adenovirus vector. In someembodiments, the adenovirus may be a high-cloning capacity or “gutless”adenovirus, where all coding viral regions apart from the 5′ and 3′inverted terminal repeats (ITRs) and the packaging signal (‘I’) aredeleted from the virus to increase its packaging capacity. In yet otherembodiments, the viral vector may be an HSV-1 vector. In someembodiments, the HSV-1-based vector is helper dependent, and in otherembodiments it is helper independent. For example, an amplicon vectorthat retains only the packaging sequence requires a helper virus withstructural components for packaging, while a 30 kb-deleted HSV-1 vectorthat removes non-essential viral functions does not require helpervirus. In additional embodiments, the viral vector may be bacteriophageT4. In some embodiments, the bacteriophage T4 may be able to package anylinear or circular DNA or RNA molecules when the head of the virus isemptied. In further embodiments, the viral vector may be a baculovirusvector. In yet further embodiments, the viral vector may be a retrovirusvector. In embodiments using AAV or lentiviral vectors, which havesmaller cloning capacity, it may be necessary to use more than onevector to deliver all the components of a vector system as disclosedherein. For example, one AAV vector may contain sequences encoding anRNA-guided DNA nuclease such as a Cas nuclease, while a second AAVvector may contain one or more guide sequences.

In some embodiments, the vector may be capable of driving expression ofone or more coding sequences in a cell. In some embodiments, the cellmay be a prokaryotic cell, such as, e.g., a bacterial cell. In someembodiments, the cell may be a eukaryotic cell, such as, e.g., a yeast,plant, insect, or mammalian cell. In some embodiments, the eukaryoticcell may be a mammalian cell. In some embodiments, the eukaryotic cellmay be a rodent cell. In some embodiments, the eukaryotic cell may be ahuman cell. Suitable promoters to drive expression in different types ofcells are known in the art. In some embodiments, the promoter may bewild type. In other embodiments, the promoter may be modified for moreefficient or efficacious expression. In yet other embodiments, thepromoter may be truncated yet retain its function. For example, thepromoter may have a normal size or a reduced size that is suitable forproper packaging of the vector into a virus.

In some embodiments, the vector may comprise a nucleotide sequenceencoding an RNA-guided DNA nuclease such as a nuclease described herein.In some embodiments, the nuclease encoded by the vector may be a Casprotein. In some embodiments, the vector system may comprise one copy ofthe nucleotide sequence encoding the nuclease. In other embodiments, thevector system may comprise more than one copy of the nucleotide sequenceencoding the nuclease. In some embodiments, the nucleotide sequenceencoding the nuclease may be operably linked to at least onetranscriptional or translational control sequence. In some embodiments,the nucleotide sequence encoding the nuclease may be operably linked toat least one promoter.

In some embodiments, the promoter may be constitutive, inducible, ortissue-specific. In some embodiments, the promoter may be a constitutivepromoter. Non-limiting exemplary constitutive promoters includecytomegalovirus immediate early promoter (CMV), simian virus (SV40)promoter, adenovirus major late (MLP) promoter, Rous sarcoma virus (RSV)promoter, mouse mammary tumor virus (MMTV) promoter, phosphoglyceratekinase (PGK) promoter, elongation factor-alpha (EF1a) promoter,ubiquitin promoters, actin promoters, tubulin promoters, immunoglobulinpromoters, a functional fragment thereof, or a combination of any of theforegoing. In some embodiments, the promoter may be a CMV promoter. Insome embodiments, the promoter may be a truncated CMV promoter. In otherembodiments, the promoter may be an EF1a promoter. In some embodiments,the promoter may be an inducible promoter. Non-limiting exemplaryinducible promoters include those inducible by heat shock, light,chemicals, peptides, metals, steroids, antibiotics, or alcohol. In someembodiments, the inducible promoter may be one that has a low basal(non-induced) expression level, such as, e.g., the Tet-On® promoter(Clontech).

In some embodiments, the promoter may be a tissue-specific promoter,e.g., a promoter specific for expression in the liver.

The vector may further comprise a nucleotide sequence encoding the guideRNA described herein. In some embodiments, the vector comprises one copyof the guide RNA. In other embodiments, the vector comprises more thanone copy of the guide RNA. In embodiments with more than one guide RNA,the guide RNAs may be non-identical such that they target differenttarget sequences, or may be identical in that they target the sametarget sequence. In some embodiments where the vectors comprise morethan one guide RNA, each guide RNA may have other different properties,such as activity or stability within a complex with an RNA-guided DNAnuclease, such as a Cas RNP complex. In some embodiments, the nucleotidesequence encoding the guide RNA may be operably linked to at least onetranscriptional or translational control sequence, such as a promoter, a3′ UTR, or a 5′ UTR. In one embodiment, the promoter may be a tRNApromoter, e.g., tRNA^(Lys3), or a tRNA chimera. See Mefferd et al., RNA.2015 21:1683-9; Scherer et al., Nucleic Acids Res. 2007 35: 2620-2628.In some embodiments, the promoter may be recognized by RNA polymeraseIII (Pol III). Non-limiting examples of Pol III promoters include U6 andH1 promoters. In some embodiments, the nucleotide sequence encoding theguide RNA may be operably linked to a mouse or human U6 promoter. Inother embodiments, the nucleotide sequence encoding the guide RNA may beoperably linked to a mouse or human H1 promoter. In embodiments withmore than one guide RNA, the promoters used to drive expression may bethe same or different. In some embodiments, the nucleotide encoding thecrRNA of the guide RNA and the nucleotide encoding the trRNA of theguide RNA may be provided on the same vector. In some embodiments, thenucleotide encoding the crRNA and the nucleotide encoding the trRNA maybe driven by the same promoter. In some embodiments, the crRNA and trRNAmay be transcribed into a single transcript. For example, the crRNA andtrRNA may be processed from the single transcript to form adouble-molecule guide RNA. Alternatively, the crRNA and trRNA may betranscribed into a single-molecule guide RNA (sgRNA). In otherembodiments, the crRNA and the trRNA may be driven by theircorresponding promoters on the same vector. In yet other embodiments,the crRNA and the trRNA may be encoded by different vectors.

In some embodiments, the nucleotide sequence encoding the guide RNA maybe located on the same vector comprising the nucleotide sequenceencoding an RNA-guided DNA nuclease such as a Cas nuclease. In someembodiments, expression of the guide RNA and of the RNA-guided DNAnuclease such as a Cas protein may be driven by their own correspondingpromoters. In some embodiments, expression of the guide RNA may bedriven by the same promoter that drives expression of the RNA-guided DNAnuclease such as a Cas protein. In some embodiments, the guide RNA andthe RNA-guided DNA nuclease such as a Cas protein transcript may becontained within a single transcript. For example, the guide RNA may bewithin an untranslated region (UTR) of the RNA-guided DNA nuclease suchas a Cas protein transcript. In some embodiments, the guide RNA may bewithin the 5′ UTR of the transcript. In other embodiments, the guide RNAmay be within the 3′ UTR of the transcript. In some embodiments, theintracellular half-life of the transcript may be reduced by containingthe guide RNA within its 3′ UTR and thereby shortening the length of its3′ UTR. In additional embodiments, the guide RNA may be within an intronof the transcript. In some embodiments, suitable splice sites may beadded at the intron within which the guide RNA is located such that theguide RNA is properly spliced out of the transcript. In someembodiments, expression of the RNA-guided DNA nuclease such as a Casprotein and the guide RNA from the same vector in close temporalproximity may facilitate more efficient formation of the CRISPR RNPcomplex.

In some embodiments, the compositions comprise a vector system. In someembodiments, the vector system may comprise one single vector. In otherembodiments, the vector system may comprise two vectors. In additionalembodiments, the vector system may comprise three vectors. Whendifferent guide RNAs are used for multiplexing, or when multiple copiesof the guide RNA are used, the vector system may comprise more thanthree vectors.

In some embodiments, the vector system may comprise inducible promotersto start expression only after it is delivered to a target cell.Non-limiting exemplary inducible promoters include those inducible byheat shock, light, chemicals, peptides, metals, steroids, antibiotics,or alcohol. In some embodiments, the inducible promoter may be one thathas a low basal (non-induced) expression level, such as, e.g., theTet-On® promoter (Clontech).

In additional embodiments, the vector system may comprisetissue-specific promoters to start expression only after it is deliveredinto a specific tissue.

The vector may be delivered by liposome, a nanoparticle, an exosome, ora microvesicle. The vector may also be delivered by a lipid nanoparticle(LNP); see e.g., U.S. Ser. No. 62/433,228, filed Dec. 12, 2016 andentitled “LIPID NANOPARTICLE FORMULATIONS FOR CRISPR/CAS COMPONENTS,”the contents of which are hereby incorporated by reference in theirentirety. Any of the LNPs and LNP formulations described herein aresuitable for delivery of the guides alone or together a cas nuclease oran mRNA encoding a cas nuclease. In some embodiments, an LNP compositionis encompassed comprising: an RNA component and a lipid component,wherein the lipid component comprises an amine lipid, a neutral lipid, ahelper lipid, and a stealth lipid; and wherein the N/P ratio is about1-10.

In some instances, the lipid component comprises Lipid A or its acetalanalog, cholesterol, DSPC, and PEG-DMG; and wherein the N/P ratio isabout 1-10. In some embodiments, the lipid component comprises: about40-60 mol-% amine lipid; about 5-15 mol-% neutral lipid; and about1.5-10 mol-% PEG lipid, wherein the remainder of the lipid component ishelper lipid, and wherein the N/P ratio of the LNP composition is about3-10. In some embodiments, the lipid component comprises about 50-60mol-% amine lipid; about 8-10 mol-% neutral lipid; and about 2.5-4 mol-%PEG lipid, wherein the remainder of the lipid component is helper lipid,and wherein the N/P ratio of the LNP composition is about 3-8. In someinstances, the lipid component comprises: about 50-60 mol-% amine lipid;about 5-15 mol-% DSPC; and about 2.5-4 mol-% PEG lipid, wherein theremainder of the lipid component is cholesterol, and wherein the N/Pratio of the LNP composition is about 3-8. In some instances, the lipidcomponent comprises: 48-53 mol-% Lipid A; about 8-10 mol-% DSPC; and1.5-10 mol-% PEG lipid, wherein the remainder of the lipid component ischolesterol, and wherein the N/P ratio of the LNP composition is3-8±0.2.

In some embodiments, the vector may be delivered systemically. In someembodiments, the vector may be delivered into the hepatic circulation.

This description and exemplary embodiments should not be taken aslimiting. For the purposes of this specification and appended claims,unless otherwise indicated, all numbers expressing quantities,percentages, or proportions, and other numerical values used in thespecification and claims, are to be understood as being modified in allinstances by the term “about,” to the extent they are not already somodified. Accordingly, unless indicated to the contrary, the numericalparameters set forth in the following specification and attached claimsare approximations that may vary depending upon the desired propertiessought to be obtained. At the very least, and not as an attempt to limitthe application of the doctrine of equivalents to the scope of theclaims, each numerical parameter should at least be construed in lightof the number of reported significant digits and by applying ordinaryrounding techniques.

It is noted that, as used in this specification and the appended claims,the singular forms “a,” “an,” and “the,” and any singular use of anyword, include plural referents unless expressly and unequivocallylimited to one referent. As used herein, the term “include” and itsgrammatical variants are intended to be non-limiting, such thatrecitation of items in a list is not to the exclusion of other likeitems that can be substituted or added to the listed items.

Examples

The following examples are provided to illustrate certain disclosedembodiments and are not to be construed as limiting the scope of thisdisclosure in any way.

Example 1. Materials and Methods

In Vitro Transcription (“IVT”) of Nuclease mRNA

Capped and polyadenylated Streptococcus pyogenes (“Spy”) Cas9 mRNAcontaining N1-methyl pseudo-U was generated by in vitro transcriptionusing a linearized plasmid DNA template and T7 RNA polymerase. PlasmidDNA containing a T7 promoter, a sequence for transcription according toSEQ ID NO: 1 or 2, and a 100 nt poly (A/T) region was linearized byincubating at 37° C. for 2 hours with XbaI with the followingconditions: 200 ng/μL plasmid, 2 U/μL XbaI (NEB), and 1× reactionbuffer. The XbaI was inactivated by heating the reaction at 65° C. for20 min. The linearized plasmid was purified from enzyme and buffer saltsusing a silica maxi spin column (Epoch Life Sciences) and analyzed byagarose gel to confirm linearization. The IVT reaction to generate Cas9modified mRNA was incubated at 37° C. for 4 hours in the followingconditions: 50 ng/μL linearized plasmid; 2 mM each of GTP, ATP, CTP, andN1-methyl pseudo-UTP (Trilink); 10 mM ARCA (Trilink); 5 U/μL T7 RNApolymerase (NEB); 1 U/μL Murine RNase inhibitor (NEB); 0.004 U/μLInorganic E. coli pyrophosphatase (NEB); and 1× reaction buffer. Afterthe 4-hour incubation, TURBO DNase (ThermoFisher) was added to a finalconcentration of 0.01 U/μL, and the reaction was incubated for anadditional 30 minutes to remove the DNA template. The Cas9 mRNA waspurified from enzyme and nucleotides using a MegaClear TranscriptionClean-up kit per the manufacturer's protocol (ThermoFisher).Alternatively, the mRNA was purified through a precipitation protocol,which in some cases was followed by HPLC-based purification. Briefly,after the DNase digestion, the mRNA was precipitated by adding 0.21× volof a 7.5 M LiCl solution and mixing, and the precipitated mRNA waspelleted by centrifugation. Once the supernatant was removed, the mRNAwas reconstituted in water. The mRNA was precipitated again usingammonium acetate and ethanol. 5M Ammonium acetate was added to the mRNAsolution for a final concentration of 2M along with 2× volume of 100%EtOH. The solution was mixed and incubated at −20° C. for 15 min. Theprecipitated mRNA was again pelleted by centrifugation, the supernatantwas removed, and the mRNA was reconstituted in water. As a final step,the mRNA was precipitated using sodium acetate and ethanol. 1/10 volumeof 3 M sodium acetate (pH 5.5) was added to the solution along with 2×volume of 100% EtOH. The solution was mixed and incubated at −20° C. for15 min. The precipitated mRNA was again pelleted by centrifugation, thesupernatant was removed, the pellet was washed with 70% cold ethanol andallowed to air dry. The mRNA was reconstituted in water. For HPLCpurified mRNA, after the LiCl precipitation and reconstitution, the mRNAwas purified by RP-IP HPLC (see, e.g., Kariko, et al. Nucleic AcidsResearch, 2011, Vol. 39, No. 21 e142). The fractions chosen for poolingwere combined and desalted by sodium acetate/ethanol precipitation asdescribed above. The transcript concentration was determined bymeasuring the light absorbance at 260 nm (Nanodrop), and the transcriptwas analyzed by capillary electrophoresis by Bioanlayzer (Agilent).

When SEQ ID NOs: 1 and 2 are referred to below with respect to RNAs, itis understood that Ts should be replaced with Us (which were N1-methylpseudouridines as described above). Cas9 mRNAs used in the Examplesinclude a 5′ cap and a 3′ poly-A tail, e.g., up to 100 nts, and areidentified by SEQ ID NO.

SEQ ID NO: 1: Cas9 sequence 1 for transcription.GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGTGTGTCGTTGCAGGCCTTATTCGGATCCGCCACCATGGACAAGAAGTACAGCATCGGACTGGACATCGGAACAAACAGCGTCGGATGGGCAGTCATCACAGACGAATACAAGGTCCCGAGCAAGAAGTTCAAGGTCCTGGGAAACACAGACAGACACAGCATCAAGAAGAACCTGATCGGAGCACTGCTGTTCGACAGCGGAGAAACAGCAGAAGCAACAAGACTGAAGAGAACAGCAAGAAGAAGATACACAAGAAGAAAGAACAGAATCTGCTACCTGCAGGAAATCTTCAGCAACGAAATGGCAAAGGTCGACGACAGCTTCTTCCACAGACTGGAAGAAAGCTTCCTGGTCGAAGAAGACAAGAAGCACGAAAGACACCCGATCTTCGGAAACATCGTCGACGAAGTCGCATACCACGAAAAGTACCCGACAATCTACCACCTGAGAAAGAAGCTGGTCGACAGCACAGACAAGGCAGACCTGAGACTGATCTACCTGGCACTGGCACACATGATCAAGTTCAGAGGACACTTCCTGATCGAAGGAGACCTGAACCCGGACAACAGCGACGTCGACAAGCTGTTCATCCAGCTGGTCCAGACATACAACCAGCTGTTCGAAGAAAACCCGATCAACGCAAGCGGAGTCGACGCAAAGGCAATCCTGAGCGCAAGACTGAGCAAGAGCAGAAGACTGGAAAACCTGATCGCACAGCTGCCGGGAGAAAAGAAGAACGGACTGTTCGGAAACCTGATCGCACTGAGCCTGGGACTGACACCGAACTTCAAGAGCAACTTCGACCTGGCAGAAGACGCAAAGCTGCAGCTGAGCAAGGACACATACGACGACGACCTGGACAACCTGCTGGCACAGATCGGAGACCAGTACGCAGACCTGTTCCTGGCAGCAAAGAACCTGAGCGACGCAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAAATCACAAAGGCACCGCTGAGCGCAAGCATGATCAAGAGATACGACGAACACCACCAGGACCTGACACTGCTGAAGGCACTGGTCAGACAGCAGCTGCCGGAAAAGTACAAGGAAATCTTCTTCGACCAGAGCAAGAACGGATACGCAGGATACATCGACGGAGGAGCAAGCCAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAAGATGGACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAAGACCTGCTGAGAAAGCAGAGAACATTCGACAACGGAAGCATCCCGCACCAGATCCACCTGGGAGAACTGCACGCAATCCTGAGAAGACAGGAAGACTTCTACCCGTTCCTGAAGGACAACAGAGAAAAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTACGTCGGACCGCTGGCAAGAGGAAACAGCAGATTCGCATGGATGACAAGAAAGAGCGAAGAAACAATCACACCGTGGAACTTCGAAGAAGTCGTCGACAAGGGAGCAAGCGCACAGAGCTTCATCGAAAGAATGACAAACTTCGACAAGAACCTGCCGAACGAAAAGGTCCTGCCGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTACAACGAACTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCCGGCATTCCTGAGCGGAGAACAGAAGAAGGCAATCGTCGACCTGCTGTTCAAGACAAACAGAAAGGTCACAGTCAAGCAGCTGAAGGAAGACTACTTCAAGAAGATCGAATGCTTCGACAGCGTCGAAATCAGCGGAGTCGAAGACAGATTCAACGCAAGCCTGGGAACATACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGACAACGAAGAAAACGAAGACATCCTGGAAGACATCGTCCTGACACTGACACTGTTCGAAGACAGAGAAATGATCGAAGAAAGACTGAAGACATACGCACACCTGTTCGACGACAAGGTCATGAAGCAGCTGAAGAGAAGAAGATACACAGGATGGGGAAGACTGAGCAGAAAGCTGATCAACGGAATCAGAGACAAGCAGAGCGGAAAGACAATCCTGGACTTCCTGAAGAGCGACGGATTCGCAAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACATTCAAGGAAGACATCCAGAAGGCACAGGTCAGCGGACAGGGAGACAGCCTGCACGAACACATCGCAAACCTGGCAGGAAGCCCGGCAATCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAACTGGTCAAGGTCATGGGAAGACACAAGCCGGAAAACATCGTCATCGAAATGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAATGAAGAGAATCGAAGAAGGAATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACACCCGGTCGAAAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCTGCAGAACGGAAGAGACATGTACGTCGACCAGGAACTGGACATCAACAGACTGAGCGACTACGACGTCGACCACATCGTCCCGCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTCCTGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGTCCCGAGCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGACAGCTGCTGAACGCAAAGCTGATCACACAGAGAAAGTTCGACAACCTGACAAAGGCAGAGAGAGGAGGACTGAGCGAACTGGACAAGGCAGGATTCATCAAGAGACAGCTGGTCGAAACAAGACAGATCACAAAGCACGTCGCACAGATCCTGGACAGCAGAATGAACACAAAGTACGACGAAAACGACAAGCTGATCAGAGAAGTCAAGGTCATCACACTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGACTTCCAGTTCTACAAGGTCAGAGAAATCAACAACTACCACCACGCACACGACGCATACCTGAACGCAGTCGTCGGAACAGCACTGATCAAGAAGTACCCGAAGCTGGAAAGCGAATTCGTCTACGGAGACTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGAGCGAACAGGAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAAGACAGAAATCACACTGGCAAACGGAGAAATCAGAAAGAGACCGCTGATCGAAACAAACGGAGAAACAGGAGAAATCGTCTGGGACAAGGGAAGAGACTTCGCAACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCGTCAAGAAGACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAGCATCCTGCCGAAGAGAAACAGCGACAAGCTGATCGCAAGAAAGAAGGACTGGGACCCGAAGAAGTACGGAGGATTCGACAGCCCGACAGTCGCATACAGCGTCCTGGTCGTCGCAAAGGTCGAAAAGGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGCTGGGAATCACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGATCGACTTCCTGGAAGCAAAGGGATACAAGGAAGTCAAGAAGGACCTGATCATCAAGCTGCCGAAGTACAGCCTGTTCGAACTGGAAAACGGAAGAAAGAGAATGCTGGCAAGCGCAGGAGAACTGCAGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGTCAACTTCCTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCTGTTCGTCGAACAGCACAAGCACTACCTGGACGAAATCATCGAACAGATCAGCGAATTCAGCAAGAGAGTCATCCTGGCAGACGCAAACCTGGACAAGGTCCTGAGCGCATACAACAAGCACAGAGACAAGCCGATCAGAGAACAGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTGGGAGCACCGGCAGCATTCAAGTACTTCGACACAACAATCGACAGAAAGAGATACACAAGCACAAAGGAAGTCCTGGACGCAACACTGATCCACCAGAGCATCACAGGACTGTACGAAACAAGAATCGACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAAGCCCGAAGAAGAAGAGAAAGGTCTAGCTAGCCATCACATTTAAAAGCATCTCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAATAGCTTATTCATCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAACATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAATAAAAAATGGAA AGAACCTCGAGSEQ ID NO: 2: Cas9 sequence 2 for transcription.GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGTGTGTCGTTGCAGGCCTTATTCGGATCCATGGATAAGAAGTACTCAATCGGGCTGGATATCGGAACTAATTCCGTGGGTTGGGCAGTGATCACGGATGAATACAAAGTGCCGTCCAAGAAGTTCAAGGTCCTGGGGAACACCGATAGACACAGCATCAAGAAAAATCTCATCGGAGCCCTGCTGTTTGACTCCGGCGAAACCGCAGAAGCGACCCGGCTCAAACGTACCGCGAGGCGACGCTACACCCGGCGGAAGAATCGCATCTGCTATCTGCAAGAGATCTTTTCGAACGAAATGGCAAAGGTCGACGACAGCTTCTTCCACCGCCTGGAAGAATCTTTCCTGGTGGAGGAGGACAAGAAGCATGAACGGCATCCTATCTTTGGAAACATCGTCGACGAAGTGGCGTACCACGAAAAGTACCCGACCATCTACCATCTGCGGAAGAAGTTGGTTGACTCAACTGACAAGGCCGACCTCAGATTGATCTACTTGGCCCTCGCCCATATGATCAAATTCCGCGGACACTTCCTGATCGAAGGCGATCTGAACCCTGATAACTCCGACGTGGATAAGCTTTTCATTCAACTGGTGCAGACCTACAACCAACTGTTCGAAGAAAACCCAATCAATGCTAGCGGCGTCGATGCCAAGGCCATCCTGTCCGCCCGGCTGTCGAAGTCGCGGCGCCTCGAAAACCTGATCGCACAGCTGCCGGGAGAGAAAAAGAACGGACTTTTCGGCAACTTGATCGCTCTCTCACTGGGACTCACTCCCAATTTCAAGTCCAATTTTGACCTGGCCGAGGACGCGAAGCTGCAACTCTCAAAGGACACCTACGACGACGACTTGGACAATTTGCTGGCACAAATTGGCGATCAGTACGCGGATCTGTTCCTTGCCGCTAAGAACCTTTCGGACGCAATCTTGCTGTCCGATATCCTGCGCGTGAACACCGAAATAACCAAAGCGCCGCTTAGCGCCTCGATGATTAAGCGGTACGACGAGCATCACCAGGATCTCACGCTGCTCAAAGCGCTCGTGAGACAGCAACTGCCTGAAAAGTACAAGGAGATCTTCTTCGACCAGTCCAAGAATGGGTACGCAGGGTACATCGATGGAGGCGCTAGCCAGGAAGAGTTCTATAAGTTCATCAAGCCAATCCTGGAAAAGATGGACGGAACCGAAGAACTGCTGGTCAAGCTGAACAGGGAGGATCTGCTCCGGAAACAGAGAACCTTTGACAACGGATCCATTCCCCACCAGATCCATCTGGGTGAGCTGCACGCCATCTTGCGGCGCCAGGAGGACTTTTACCCATTCCTCAAGGACAACCGGGAAAAGATCGAGAAAATTCTGACGTTCCGCATCCCGTATTACGTGGGCCCACTGGCGCGCGGCAATTCGCGCTTCGCGTGGATGACTAGAAAATCAGAGGAAACCATCACTCCTTGGAATTTCGAGGAAGTTGTGGATAAGGGAGCTTCGGCACAAAGCTTCATCGAACGAATGACCAACTTCGACAAGAATCTCCCAAACGAGAAGGTGCTTCCTAAGCACAGCCTCCTTTACGAATACTTCACTGTCTACAACGAACTGACTAAAGTGAAATACGTTACTGAAGGAATGAGGAAGCCGGCCTTTCTGTCCGGAGAACAGAAGAAAGCAATTGTCGATCTGCTGTTCAAGACCAACCGCAAGGTGACCGTCAAGCAGCTTAAAGAGGACTACTTCAAGAAGATCGAGTGTTTCGACTCAGTGGAAATCAGCGGGGTGGAGGACAGATTCAACGCTTCGCTGGGAACCTATCATGATCTCCTGAAGATCATCAAGGACAAGGACTTCCTTGACAACGAGGAGAACGAGGACATCCTGGAAGATATCGTCCTGACCTTGACCCTTTTCGAGGATCGCGAGATGATCGAGGAGAGGCTTAAGACCTACGCTCATCTCTTCGACGATAAGGTCATGAAACAACTCAAGCGCCGCCGGTACACTGGTTGGGGCCGCCTCTCCCGCAAGCTGATCAACGGTATTCGCGATAAACAGAGCGGTAAAACTATCCTGGATTTCCTCAAATCGGATGGCTTCGCTAATCGTAACTTCATGCAATTGATCCACGACGACAGCCTGACCTTTAAGGAGGACATCCAAAAAGCACAAGTGTCCGGACAGGGAGACTCACTCCATGAACACATCGCGAATCTGGCCGGTTCGCCGGCGATTAAGAAGGGAATTCTGCAAACTGTGAAGGTGGTCGACGAGCTGGTGAAGGTCATGGGACGGCACAAACCGGAGAATATCGTGATTGAAATGGCCCGAGAAAACCAGACTACCCAGAAGGGCCAGAAAAACTCCCGCGAAAGGATGAAGCGGATCGAAGAAGGAATCAAGGAGCTGGGCAGCCAGATCCTGAAAGAGCACCCGGTGGAAAACACGCAGCTGCAGAACGAGAAGCTCTACCTGTACTATTTGCAAAATGGACGGGACATGTACGTGGACCAAGAGCTGGACATCAATCGGTTGTCTGATTACGACGTGGACCACATCGTTCCACAGTCCTTTCTGAAGGATGACTCGATCGATAACAAGGTGTTGACTCGCAGCGACAAGAACAGAGGGAAGTCAGATAATGTGCCATCGGAGGAGGTCGTGAAGAAGATGAAGAATTACTGGCGGCAGCTCCTGAATGCGAAGCTGATTACCCAGAGAAAGTTTGACAATCTCACTAAAGCCGAGCGCGGCGGACTCTCAGAGCTGGATAAGGCTGGATTCATCAAACGGCAGCTGGTCGAGACTCGGCAGATTACCAAGCACGTGGCGCAGATCTTGGACTCCCGCATGAACACTAAATACGACGAGAACGATAAGCTCATCCGGGAAGTGAAGGTGATTACCCTGAAAAGCAAACTTGTGTCGGACTTTCGGAAGGACTTTCAGTTTTACAAAGTGAGAGAAATCAACAACTACCATCACGCGCATGACGCATACCTCAACGCTGTGGTCGGTACCGCCCTGATCAAAAAGTACCCTAAACTTGAATCGGAGTTTGTGTACGGAGACTACAAGGTCTACGACGTGAGGAAGATGATAGCCAAGTCCGAACAGGAAATCGGGAAAGCAACTGCGAAATACTTCTTTTACTCAAACATCATGAACTTTTTCAAGACTGAAATTACGCTGGCCAATGGAGAAATCAGGAAGAGGCCACTGATCGAAACTAACGGAGAAACGGGCGAAATCGTGTGGGACAAGGGCAGGGACTTCGCAACTGTTCGCAAAGTGCTCTCTATGCCGCAAGTCAATATTGTGAAGAAAACCGAAGTGCAAACCGGCGGATTTTCAAAGGAATCGATCCTCCCAAAGAGAAATAGCGACAAGCTCATTGCACGCAAGAAAGACTGGGACCCGAAGAAGTACGGAGGATTCGATTCGCCGACTGTCGCATACTCCGTCCTCGTGGTGGCCAAGGTGGAGAAGGGAAAGAGCAAAAAGCTCAAATCCGTCAAAGAGCTGCTGGGGATTACCATCATGGAACGATCCTCGTTCGAGAAGAACCCGATTGATTTCCTCGAGGCGAAGGGTTACAAGGAGGTGAAGAAGGATCTGATCATCAAACTCCCCAAGTACTCACTGTTCGAACTGGAAAATGGTCGGAAGCGCATGCTGGCTTCGGCCGGAGAACTCCAAAAAGGAAATGAGCTGGCCTTGCCTAGCAAGTACGTCAACTTCCTCTATCTTGCTTCGCACTACGAAAAACTCAAAGGGTCACCGGAAGATAACGAACAGAAGCAGCTTTTCGTGGAGCAGCACAAGCATTATCTGGATGAAATCATCGAACAAATCTCCGAGTTTTCAAAGCGCGTGATCCTCGCCGACGCCAACCTCGACAAAGTCCTGTCGGCCTACAATAAGCATAGAGATAAGCCGATCAGAGAACAGGCCGAGAACATTATCCACTTGTTCACCCTGACTAACCTGGGAGCCCCAGCCGCCTTCAAGTACTTCGATACTACTATCGATCGCAAAAGATACACGTCCACCAAGGAAGTTCTGGACGCGACCCTGATCCACCAAAGCATCACTGGACTCTACGAAACTAGGATCGATCTGTCGCAGCTGGGTGGCGATGGCGGTGGATCTCCGAAAAAGAAGAGAAAGGTGTAATGAGCTAGCCATCACATTTAAAAGCATCTCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAATAGCTTATTCATCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAACATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAATAAAAAATGGAAAG AACCTCGAGHuman TTR Guide Design and Human TTR with Cynomolgus Monkey HomologyGuide Design

Initial guide selection was performed in silico using a human referencegenome (e.g., hg38) and user defined genomic regions of interest (e.g.,TTR protein coding exons), for identifying PAMs in the regions ofinterest. For each identified PAM, analyses were performed andstatistics reported. gRNA molecules were further selected and rankordered based on a number of criteria (e.g., GC content, predictedon-target activity, and potential off-target activity).

A total of 68 guide RNAs were designed toward TTR (ENSG00000118271)targeting the protein coding regions within Exon 1, 2, 3 and 4. Of thetotal 68 guides, 33 were 100% homologous in cynomolgus monkey (“cyno”).In addition, for 10 of the human TTR guides which were not perfectlyhomologous in cyno, “surrogate” guides were designed and made inparallel to perfectly match the corresponding cyno target sequence.These “surrogate” or “tool” guides may be screened in cyno, e.g., toapproximate the activity and function of the homologous human guidesequence. Guide sequences and corresponding genomic coordinates areprovided (Table 1). All of the guide RNAs were made as dual guide RNAs,and a subset of the guide sequences were made as modified single guideRNA (Table 2). Guide ID alignment across dual guide RNA (dgRNA) IDs,modified single guide RNA (sgRNA) IDs, the number of mismatches to thecyno genome as well as the cyno exact matched IDs are provided (Table3). Where dgRNAs are used in the experiments detailed throughout theExamples, SEQ ID NO: 270 was used.Cas9 mRNA and Guide RNA Delivery In Vitro

HEK293_Cas9 Cell Line.

The human embryonic kidney adenocarcinoma cell line HEK293constitutively expressing Spy Cas9 (“HEK293_Cas9”) was cultured in DMEMmedia supplemented with 10% fetal bovine serum and 500 μg/ml G418. Cellswere plated at a density of 10,000 cells/well in a 96-well plate 24hours prior to transfection. Cells were transfected with LipofectamineRNAiMAX (ThermoFisher, Cat. 13778150) per the manufacturer's protocol.Cells were transfected with a lipoplex containing individual crRNA (25nM), trRNA (25 nM), Lipofectamine RNAiMAX (0.3 μL/well) and OptiMem.

HUH7 Cell Line.

The human hepatocellular carcinoma cell line HUH7 (Japanese Collectionof Research Bioresources Cell Bank, Cat. JCRB0403) was cultured in DMEMmedia supplemented with 10% fetal bovine serum. Cells were plated on ata density of 15,000 cells/well in a 96-well plate 20 hours prior totransfection. Cells were transfected with Lipofectamine MessengerMAX(ThermoFisher, Cat. LMRNA003) per the manufacturer's protocol. Cellswere sequentially transfected with a lipoplex containing Spy Cas9 mRNA(100 ng), MessengerMAX (0.3 μL/well) and OptiMem followed by a separatelipoplex containing individual crRNA (25 nM), tracer RNA (25 nM),MessengerMAX (0.3 μL/well) and OptiMem.

HepG2 Cell Line.

The human hepatocellular carcinoma cell line HepG2 (American TypeCulture Collection, Cat. HB-8065) was cultured in DMEM mediasupplemented with 10% fetal bovine serum. Cells were counted and platedon Bio-coat collagen I coated 96-well plates (ThermoFisher, Cat. 877272)at a density of 10,000 cells/well in a 96-well plate 24 hours prior totransfection. Cells were transfected with Lipofectamine 2000(ThermoFisher, Cat. 11668019) per the manufacturer's protocol. Cellswere sequentially transfected with lipoplex containing Spy Cas9 mRNA(100 ng), Lipofectamine 2000 (0.2 μL/well) and OptiMem followed by aseparate lipoplex containing individual crRNA (25 nM), tracer RNA (25nM), Lipofectamine 2000 (0.2 μL/well) and OptiMem.

Primary Liver Hepatocytes.

Primary human liver hepatocytes (PHH) and primary cynomolgus liverhepatocytes (PCH) (Gibco) were cultured per the manufacturer's protocol(Invitrogen, protocol 11.28.2012). In brief, the cells were thawed andresuspended in hepatocyte thawing medium with supplements (Gibco, Cat.CM7000) followed by centrifugation at 100 g for 10 minutes for human and80 g for 4 minutes for cyno. The supernatant was discarded and thepelleted cells resuspended in hepatocyte plating medium plus supplementpack (Invitrogen, Cat. A1217601 and CM3000). Cells were counted andplated on Bio-coat collagen I coated 96-well plates (ThermoFisher, Cat.877272) at a density of 33,000 cells/well for human or 60,000 cells/wellfor cyno (or 65,000 cells/well when assaying effects on TTR protein,described further below). Plated cells were allowed to settle and adherefor 6 or 24 hours in a tissue culture incubator at 37° C. and 5% CO₂atmosphere. After incubation cells were checked for monolayer formationand media was replaced with hepatocyte culture medium with serum-freesupplement pack (Invitrogen, Cat. A1217601 and CM4000).

Lipofectamine RNAiMax (ThermoFisher, Cat. 13778150) based transfectionswere conducted as per the manufacturer's protocol. Cells weresequentially transfected with a lipoplex containing Spy Cas9 mRNA (100ng), Lipofectamine RNAiMax (0.4 μL/well) and OptiMem followed by aseparate lipoplex containing crRNA (25 nM) and tracer RNA (25 nM) orsgRNA (25 nM), Lipofectamine RNAiMax (0.4 μL/well) and OptiMem.

Ribonucleotide formation was performed prior to electroporation ortransfection of Spy Cas9 protein loaded with guide RNAs (RNPs) ontocells. For dual guide (dgRNAs), individual crRNA and trRNA waspre-annealed by mixing equivalent amounts of reagent and incubating at95° C. for 2 min and cooling to room temperature. Single guide (sgRNAs)were boiled at 95° C. for 2 min and cooling to room temperature. Theboiled dgRNA or sgRNA was incubated with Spy Cas9 protein in Optimem for10 minutes at room temperature to form a ribonucleoprotein (RNP)complex.

For RNP electroporation into primary human and cyno hepatocytes, thecells are thawed and resuspended in Lonza electroporation Primary CellP3 buffer at a concentration of 2500 cells per μL for human hepatocytesand 3500 cells per μL for cyno hepatocytes. A volume of 20 μL ofresuspended cells and 5 μL of RNP are mixed together per guide. 20μL ofthe mixture is placed into a Lonza electroporation plate. The cells wereelectroporated using the Lonza nucleofector with the preset protocolEX-147. Post electroporation, the cells are transferred into a Biocoatplate containing pre-warmed maintenance media and placed in a tissueculture incubator at 37° C. and 5% CO₂.

For RNP lipoplex transfections, cells were transfected withLipofectamine RNAiMAX (ThermoFisher, Cat. 13778150) per themanufacturer's protocol. Cells were transfected with an RNP containingSpy Cas9 (10 nM), individual guide (10 nM), tracer RNA (10 nM),Lipofectamine RNAiMAX (1.0 μL/well) and OptiMem. RNP formation wasperformed as described above.

LNPs were formed either by microfluidic mixing of the lipid and RNAsolutions using a Precision Nanosystems NanoAssemblr™ BenchtopInstrument, per the manufacturer's protocol, or cross-flow mixing.

LNP Formulation—NanoAssemblr

In general, the lipid nanoparticle components were dissolved in 100%ethanol with the lipid component of various molar ratios. The RNA cargoswere dissolved in 25 mM citrate, 100 mM NaCl, pH 5.0, resulting in aconcentration of RNA cargo of approximately 0.45 mg/mL. The LNPs wereformulated with a lipid amine to RNA phosphate (N:P) molar ratio ofabout 4.5 or about 6, with the ratio of mRNA to gRNA at 1:1 by weight.

The LNPs were formed by microfluidic mixing of the lipid and RNAsolutions using a Precision Nanosystems NanoAssemblr™ BenchtopInstrument, according to the manufacturer's protocol. A 2:1 ratio ofaqueous to organic solvent was maintained during mixing usingdifferential flow rates. After mixing, the LNPs were collected, dilutedin water (approximately 1:1 v/v), held for 1 hour at room temperature,and further diluted with water (approximately 1:1 v/v) before finalbuffer exchange. The final buffer exchange into 50 mM Tris, 45 mM NaCl,5% (w/v) sucrose, pH 7.5 (TSS) was completed with PD-10 desaltingcolumns (GE). If required, formulations were concentrated bycentrifugation with Amicon 100 kDa centrifugal filters (Millipore). Theresulting mixture was then filtered using a 0.2 μm sterile filter. Thefinal LNP was stored at −80° C. until further use.

LNP Formulation—Cross-Flow

For LNPs prepared using the cross-flow technique, the LNPs were formedby impinging jet mixing of the lipid in ethanol with two volumes of RNAsolutions and one volume of water. The lipid in ethanol is mixed througha mixing cross with the two volumes of RNA solution. A fourth stream ofwater is mixed with the outlet stream of the cross through an inlinetee. (See WO2016010840 FIG. 2.) The LNPs were held for 1 hour at roomtemperature, and further diluted with water (approximately 1:1 v/v).Diluted LNPs were concentrated using tangential flow filtration on aflat sheet cartridge (Sartorius, 100 kD MWCO) and then buffer exchangedby diafiltration into 50 mM Tris, 45 mM NaCl, 5% (w/v) sucrose, pH 7.5(TSS). Alternatively, the final buffer exchange into TSS was completedwith PD-10 desalting columns (GE). If required, formulations wereconcentrated by centrifugation with Amicon 100 kDa centrifugal filters(Millipore). The resulting mixture was then filtered using a 0.2 μmsterile filter. The final LNP was stored at 4° C. or −80° C. untilfurther use.

Formulation Analytics

Dynamic Light Scattering (“DLS”) is used to characterize thepolydispersity index (“pdi”) and size of the LNPs of the presentdisclosure. DLS measures the scattering of light that results fromsubjecting a sample to a light source. PDI, as determined from DLSmeasurements, represents the distribution of particle size (around themean particle size) in a population, with a perfectly uniform populationhaving a PDI of zero. Average particle size and polydispersity aremeasured by dynamic light scattering (DLS) using a Malvern Zetasizer DLSinstrument. LNP samples were diluted 30× in PBS prior to being measuredby DLS. Z-average diameter which is an intensity based measurement ofaverage particle size was reported along with number average diameterand pdi. A Malvern Zetasizer instrument is also used to measure the zetapotential of the LNP. Samples are diluted 1:17 (50 uL into 800 uL) in0.1×PBS, pH 7.4 prior to measurement.

A fluorescence-based assay (Ribogreen®, ThermoFisher Scientific) is usedto determine total RNA concentration and free RNA. Encapsulationefficiency is calculated as (Total RNA−Free RNA)/Total RNA. LNP samplesare diluted appropriately with 1×TE buffer containing 0.2% Triton-X 100to determine total RNA or 1×TE buffer to determine free RNA. Standardcurves are prepared by utilizing the starting RNA solution used to makethe formulations and diluted in 1×TE buffer+/−0.2% Triton-X 100. DilutedRiboGreen® dye (according to the manufacturer's instructions) is thenadded to each of the standards and samples and allowed to incubate forapproximately 10 minutes at room temperature, in the absence of light. ASpectraMax M5 Microplate Reader (Molecular Devices) is used to read thesamples with excitation, auto cutoff and emission wavelengths set to 488nm, 515 nm, and 525 nm respectively. Total RNA and free RNA aredetermined from the appropriate standard curves.

Encapsulation efficiency is calculated as (Total RNA−Free RNA)/TotalRNA. The same procedure may be used for determining the encapsulationefficiency of a DNA-based cargo component. For single-strand DNAOligreen Dye may be used, and for double-strand DNA, Picogreen Dye.

Typically, when preparing LNPs, encapsulation was >80%, particle sizewas <120 nm, and pdi was <0.2.

LNP Delivery In Vivo

Unless otherwise noted, CD-1 female mice, ranging from 6-10 weeks of agewere used in each study. Animals were weighed and grouped according tobody weight for preparing dosing solutions based on group averageweight. LNPs were dosed via the lateral tail vein in a volume of 0.2 mLper animal (approximately 10 mL per kilogram body weight). The animalswere observed at approximately 6 hours post dose for adverse effects.Body weight was measured at twenty-four hours post-administration, andanimals were euthanized at various time points by exsanguination viacardiac puncture under isoflourane anesthesia. Blood was collected intoserum separator tubes or into tubes containing buffered sodium citratefor plasma as described herein. For studies involving in vivo editing,liver tissue was collected from the median lobe or from threeindependent lobes (e.g., the right median, left median, and left laterallobes) from each animal for DNA extraction and analysis.

Transthyretin (TTR) ELISA Analysis Used in Animal Studies

Blood was collected and the serum was isolated as indicated. The totalmouse TTR serum levels were determined using a Mouse Prealbumin(Transthyretin) ELISA Kit (Aviva Systems Biology, Cat. OKIA00111); ratTTR serum levels were measured using a rat specific ELISA kit (AvivaSystems Biology catalog number OKIA00159); human TTR serum levels weremeasured using a human specific ELISA kit (Aviva Systems Biology catalognumber OKIA00081); each according to manufacture's protocol. Briefly,sera were serial diluted with kit sample diluent to a final dilution of10,000-fold, or 5,000-fold when measuring human TTR in mouse sera. 100ul of the prepared standard curve or diluted serum samples were added tothe ELISA plate, incubated for 30 minutes at room temperature thenwashed 3 times with provided wash buffer. 100 uL of detection antibodywas then added to each well and incubated for 20 minutes at roomtemperature followed by 3 washes. 100 uL of substrate is added thenincubated for 10 minutes at room temperature before the addition of 100uL stop solution. The absorbance of the contents was measured on theSpectramax M5 plate reader with analysis using SoftmaxPro version 7.0software. Serum TTR levels were quantitated off the standard curve using4 parameter logistic fit and expressed as ug/mL of serum or percentknockdown relative control (vehicle treated) animals.

Genomic DNA Isolation

Transfected cells were harvested post-transfection at 24, 48, or 72hours. The genomic DNA was extracted from each well of a 96-well plateusing 50 μL/well BuccalAmp DNA Extraction solution (Epicentre, Cat.QE09050) per manufacturer's protocol. All DNA samples were subjected toPCR and subsequent NGS analyses, as described herein.

Next-Generation Sequencing (“NGS”) Analysis

To quantitatively determine the efficiency of editing at the targetlocation in the genome, sequencing was utilized to identify the presenceof insertions and deletions introduced by gene editing.

Primers were designed around the target site within the gene of interest(e.g. TTR), and the genomic area of interest was amplified.

Additional PCR was performed per the manufacturer's protocols (Illumina)to add chemistry for sequencing. The amplicons were sequenced on anIllumina MiSeq instrument. The reads were aligned to a reference genome(e.g., the human reference genome (hg38), the cynomologus referencegenome (mf5), the rat reference genome (rn6), or the mouse referencegenome (mm10)) after eliminating those having low quality scores. Theresulting files containing the reads were mapped to the reference genome(BAM files), where reads that overlapped the target region of interestwere selected and the number of wild type reads versus the number ofreads which contain an insertion, substitution, or deletion wascalculated.

The editing percentage (e.g., the “editing efficiency” or “percentediting” or “indel frequency”) is defined as the total number ofsequence reads with insertions/deletions (“indels”) or substitutionsover the total number of sequence reads, including wild type.

Analysis of Secreted Transthyretin (“TTR”) Protein by Western Blot

Secreted levels of TTR protein in media were determined using westernblotting methods. HepG2 cells were transfected as previously describedwith select guides from Table 1. Media changes were performed every 3days post transfection. Six days post-transfection, the media wasremoved, and cells were washed once with media that did not containfetal bovine serum (FBS). Media without serum was added to the cells andincubated at 37° C. After 4 hrs the media was removed and centrifuged topellet any debris; cell number for each well was estimated based on thevalues obtained from a CTG assay on remaining cells and comparison tothe plate average. After centrifugation, the media was transferred to anew plate and stored at −20° C. An acetone precipitation of the mediawas performed to precipitate any protein that had been secreted into themedia. Four volumes of ice cold acetone were added to one volume ofmedia. The solution was mixed well and kept at −20° C. for 90 min. Theacetone:media mixture was centrifuged at 15,000×g and 4° C. for 15 min.The supernatant was discarded and the retained pellet was air dried toeliminate any residual acetone. The pellet was resuspended in 154, RIPAbuffer (Boston Bio Products, Cat. BP-115) plus freshly added proteaseinhibitor mixture consisting of complete protease inhibitor cocktail(Sigma, Cat. 11697498001) and 1 mM DTT. Lysates were mixed with Laemmlibuffer and denatured at 95° C. for 10 minutes. Western blots were runusing the NuPage system on 12% Bis-Tris gels (ThermoFisher) per themanufacturer's protocol followed by wet transfer onto 0.45 μmnitrocellulose membrane (Bio-Rad, Cat. 1620115). Blots were blockedusing 5% Dry Milk in TBS for 30 minutes on a lab rocker at roomtemperature. Blots were rinsed with TBST and probed with rabbit α-TTRmonoclonal antibody (Abcam, Cat. Ab75815) at 1:1000 in TBST. Alpha-1antitrypsin was used as a loading control (Sigma, Cat. HPA001292) at1:1000 in TBST and incubated simultaneously with the TTR primaryantibody. Blots were sealed in a bag and kept overnight at 4° C. on alab rocker. After incubation, blots were rinsed 3 times for 5 min eachin TBST and probed with secondary antibodies to Rabbit (ThermoFisher,Cat. PISA535571) at 1:25,000 in TBST for 30 min at room temperature.After incubation, blots were rinsed 3 times for 5 min each in TBST and 2times with PBS. Blots were visualized and analyzed using a Licor Odysseysystem.

Analysis of Intracellular TTR by Western Blot

The hepatocellular carcinoma cell line, HUH7, was transfected aspreviously described with select guides from Table 1. Six-dayspost-transfection, the media was removed and the cells were lysed with50 μL/well RIPA buffer (Boston Bio Products, Cat. BP-115) plus freshlyadded protease inhibitor mixture consisting of complete proteaseinhibitor cocktail (Sigma, Cat. 11697498001), 1 mM DTT, and 250 U/mlBenzonase (EMD Millipore, Cat. 71206-3). Cells were kept on ice for 30minutes at which time NaCl (1 M final concentration) was added. Celllysates were thoroughly mixed and retained on ice for 30 minutes. Thewhole cell extracts (“WCE”) were transferred to a PCR plate andcentrifuged to pellet debris. A Bradford assay (Bio-Rad, Cat. 500-0001)was used to assess protein content of the lysates. The Bradford assayprocedure was completed per the manufacturer's protocol. Extracts werestored at minus 20° C. prior to use. Western blots were performed toassess intracellular TTR protein levels. Lysates were mixed with Laemmlibuffer and denatured at 95° C. for 10 min. Western blots were run usingthe NuPage system on 12% Bis-Tris gels (ThermoFisher) per themanufacturer's protocol followed by wet transfer onto 0.45 μmnitrocellulose membrane (Bio-Rad, Cat. 1620115). After transfermembranes were rinsed thoroughly with water and stained with Ponceau Ssolution (Boston Bio Products, Cat. ST-180) to confirm complete and eventransfer. Blots were blocked using 5% Dry Milk in TBS for 30 minutes ona lab rocker at room temperature. Blots were rinsed with TBST and probedwith rabbit α-TTR monoclonal antibody (Abcam, Cat. Ab75815) at 1:1000 inTBST. (3-actin was used as a loading control (ThermoFisher, Cat. AM4302)at 1:2500 in TBST and incubated simultaneously with the TTR primaryantibody. Blots were sealed in a bag and kept overnight at 4° C. on alab rocker. After incubation, blots were rinsed 3 times for 5 minuteseach in TBST and probed with secondary antibodies to Mouse and Rabbit(ThermoFisher, Cat. PI35518 and PISA535571) at 1:25,000 each in TBST for30 min at room temperature. After incubation, blots were rinsed 3 timesfor 5 min each in TBST and 2 times with PBS. Blots were visualized andanalyzed using a Licor Odyssey system.

Example 2. Screening of dgRNA Sequences

Cross Screening of TTR dgRNAs in Multiple Cell Types

Guides in dgRNA format targeting human TTR and the cynomologus matchedsequences were delivered to HEK293_Cas9, HUH7 and HepG2 cell lines, aswell as primary human hepatocytes and primary cynomolgus monkeyhepatocytes as described in Example 1. Percent editing was determinedfor crRNAs comprising each guide sequence across each cell type and theguide sequences were then rank ordered based on highest % edit. Thescreening data for the guide sequences in Table 1 in all five cell linesare listed below (Table 4 through 11).

Table 4 shows the average and standard deviation for % Edit, % Insertion(Ins), and % Deletion (Del) for the TTR crRNAs in the human kidneyadenocarcinoma cell line, HEK293_Cas9, which constitutively overexpresses Spy Cas9 protein.

TABLE 4 TTR editing data in Hek_Cas9 cells transfected with dgRNAs StdStd Std Avg Dev Avg Dev Avg Dev % % % % % % GUIDE ID Edit Edit InsertInsert Deletion Deletion CR003335 26.59 4.73 4.73 0.65 21.87 4.09CR003336 29.09 4.57 3.31 0.24 25.78 4.32 CR003337 42.72 1.72 5.24 1.6237.48 0.70 CR003338 52.42 3.28 4.76 0.03 47.66 3.30 CR003339 56.37 4.1349.39 3.23 6.98 0.91 CR003340 42.38 8.43 27.88 4.31 14.50 4.13 CR00334120.04 5.26 6.73 1.86 13.31 3.41 CR003342 36.57 5.80 1.19 0.22 35.38 5.59CR003343 24.36 1.51 4.82 0.43 19.53 1.39 CR003344 33.87 2.93 4.32 0.5829.54 2.37 CR003345 35.02 7.05 19.00 3.58 16.01 3.48 CR003346 48.33 5.8133.03 3.12 15.30 2.72 CR003347 21.45 5.57 0.95 0.33 20.50 5.26 CR00334835.53 5.81 22.32 3.79 13.21 2.03 CR003349 13.19 4.46 8.03 2.81 5.16 1.66CR003350 22.31 4.25 5.54 0.74 16.77 3.51 CR003351 49.67 3.77 28.42 1.6921.24 2.22 CR003352 27.90 7.55 4.91 1.35 22.99 6.26 CR003353 25.03 5.163.71 0.75 21.32 4.42 CR003354 18.46 2.02 2.56 0.21 15.90 1.89 CR00335530.60 2.53 6.99 0.80 23.61 1.75 CR003356 32.21 4.71 10.03 1.39 22.193.36 CR003357 43.23 6.71 5.38 0.87 37.85 5.88 CR003358 5.44 0.86 1.290.16 4.14 0.84 CR003359 37.75 7.50 18.35 3.73 19.40 3.78 CR003360 22.683.16 2.70 0.56 19.98 2.60 CR003361 34.45 8.97 8.66 1.66 25.78 7.32CR003362 9.90 2.66 1.48 0.33 8.41 2.33 CR003363 31.03 10.74 14.77 4.2116.26 6.54 CR003364 35.65 7.90 19.17 4.24 16.48 3.76 CR003365 36.43 6.2011.83 1.88 24.61 4.45 CR003366 47.36 6.59 10.10 1.28 37.26 5.32 CR00336747.11 15.43 28.44 9.11 18.67 6.33 CR003368 40.35 10.13 3.73 0.96 36.619.17 CR003369 33.10 7.26 9.06 1.12 24.04 6.16 CR003370 34.22 5.69 4.490.67 29.73 5.06 CR003371 25.60 8.33 3.84 1.41 21.76 6.92 CR003372 15.247.92 3.25 1.61 11.99 6.31 CR003373 13.55 2.40 1.31 0.21 12.25 2.19CR003374 10.91 0.88 0.81 0.10 10.10 0.81 CR003375 11.63 3.18 0.78 0.1710.85 3.05 CR003376 28.16 4.49 1.35 0.18 26.81 4.52 CR003377 24.70 4.442.71 0.54 21.99 3.91 CR003378 20.97 2.67 4.49 0.49 16.48 2.18 CR00337926.32 2.91 5.34 0.61 20.98 2.30 CR003380 47.64 5.74 3.64 0.24 44.00 5.52CR003381 22.04 5.74 3.82 1.26 18.23 4.64 CR003382 29.95 3.13 4.46 0.4525.49 2.73 CR003383 40.47 0.64 25.12 0.45 15.35 0.66 CR003384 17.45 1.321.45 0.23 16.00 1.42 CR003385 26.19 5.62 7.36 1.57 18.82 4.06 CR00338633.12 10.65 2.94 0.63 30.18 10.03 CR003387 24.68 5.93 7.75 1.99 16.923.94 CR003388 19.23 4.41 1.41 0.39 17.82 4.07 CR003389 34.18 5.09 10.302.12 23.87 3.02 CR003390 28.02 3.77 4.31 0.25 23.71 3.61 CR003391 44.814.67 0.61 0.07 44.19 4.63 CR003392 21.67 7.52 0.85 0.26 20.82 7.27

Table 5 shows the average and standard deviation for % Edit, % Insertion(Ins), and % Deletion (Del) for the tested TTR crRNAs co-transfectedwith Spy Cas9 mRNA (SEQ ID NO:2) in the human hepatocellular carcinomacell line, HUH7.

TABLE 5 TTR editing data in HUH7 cells transfected with Spy Cas9 mRNAand dgRNAs Std Std Std Avg Dev Avg Dev Avg Dev % % % % % % GUIDE ID EditEdit Insert Insert Deletion Deletion CR003335 31.95 4.50 4.62 0.83 27.574.08 CR003336 30.05 4.25 4.14 1.07 26.56 3.55 CR003337 55.72 3.12 8.340.93 48.95 2.24 CR003338 75.64 2.03 10.22 1.42 67.06 2.79 CR003339 79.974.73 60.55 3.94 20.13 1.02 CR003340 46.93 7.12 33.33 6.01 14.23 1.65CR003341 20.58 5.98 7.78 1.64 13.20 4.44 CR003342 45.14 7.16 1.23 0.9144.66 7.68 CR003343 76.13 7.04 9.58 3.49 66.97 6.10 CR003344 64.02 3.3310.76 1.35 54.40 2.71 CR003345 72.43 2.17 41.33 0.96 32.18 1.37 CR00334618.07 1.02 13.17 1.39 6.97 3.06 CR003347 32.16 5.50 1.64 0.42 30.79 5.11CR003348 57.14 10.98 36.08 6.97 22.71 4.42 CR003349 14.14 4.99 9.73 3.264.82 1.91 CR003350 52.91 7.61 13.43 2.00 41.64 6.03 CR003351 63.51 4.6136.87 2.49 27.49 2.14 CR003352 39.68 9.53 7.62 7.42 32.79 7.37 CR00335369.18 4.59 7.73 2.46 62.87 3.13 CR003354 12.27 3.38 1.25 0.40 11.46 3.23CR003355 38.83 5.31 9.40 1.81 30.31 3.56 CR003356 49.63 5.55 18.98 2.6731.31 3.04 CR003357 36.31 5.72 6.37 1.17 30.82 4.68 CR003358 36.50 6.1710.53 1.56 26.60 4.49 CR003359 66.75 5.84 21.73 2.30 45.97 3.93 CR00336058.62 8.73 5.01 0.60 55.13 8.19 CR003361 28.68 6.52 6.84 1.26 22.44 5.31CR003362 26.43 0.83 3.43 0.32 23.76 0.85 CR003363 41.01 7.16 17.83 3.3223.78 3.97 CR003364 47.13 10.61 24.68 5.15 23.03 5.74 CR003365 60.685.25 17.77 1.57 43.82 3.73 CR003366 69.98 8.84 20.77 3.10 50.32 5.69CR003367 66.29 4.48 33.62 4.14 33.48 0.51 CR003368 31.57 11.73 3.08 0.9229.69 11.32 CR003369 24.19 6.89 7.12 2.27 17.38 4.76 CR003370 39.1611.59 4.83 1.79 35.55 10.35 CR003371 40.47 7.68 6.07 0.89 35.65 7.01CR003372 21.52 6.02 4.89 1.66 17.25 4.58 CR003373 27.29 4.45 3.31 0.6625.12 4.12 CR003374 3.10 0.68 0.45 0.24 2.87 0.54 CR003375 2.38 0.220.26 0.14 2.25 0.12 CR003376 19.42 5.60 1.37 0.45 18.55 5.28 CR00337734.93 5.47 5.59 0.88 29.89 4.71 CR003378 40.73 4.63 9.73 1.85 32.27 2.91CR003379 19.18 5.17 3.38 0.77 16.48 4.32 CR003380 31.76 5.81 3.29 0.5729.29 5.42 CR003381 99.70 0.17 1.92 0.20 99.70 0.17 CR003382 34.47 5.710.14 0.16 34.47 5.71 CR003383 42.89 10.14 2.14 0.56 41.19 9.67 CR00338417.03 1.95 0.84 0.30 16.29 1.84 CR003386 69.40 19.41 0.53 0.23 69.3419.32 CR003387 25.64 3.69 0.23 0.07 25.55 3.62 CR003388 59.48 4.29 3.880.68 56.45 4.45 CR003389 62.32 1.97 13.19 1.18 50.90 1.02 CR003390 18.974.82 3.31 0.91 16.49 3.98 CR003391 61.31 13.21 2.10 0.51 59.70 12.76CR003392 28.37 8.58 1.93 0.73 26.98 7.94

Table 6 shows the average and standard deviation for % Edit, % Insertion(Ins), and % Deletion (Del) for the tested TTR and control crRNAsco-transfected with Spy Cas9 mRNA (SEQ ID NO:2) in the humanhepatocellular carcinoma cell line, HepG2.

TABLE 6 TTR editing data in HepG2 cells transfected with Spy Cas9 mRNAand dgRNAs Std Std Std Avg Dev Avg Dev Avg Dev % % % % % % GUIDE ID EditEdit Insert Insert Deletion Deletion CR001261 49.16 7.45 16.46 3.4632.71 4.06 (control) CR001262 63.33 5.66 59.88 4.92 3.45 0.86 (control)CR001263 39.19 6.98 37.59 8.01 1.60 1.92 (control) CR001264 57.09 12.1447.47 9.25 9.61 2.89 (control) CR003335 37.19 2.12 32.96 1.67 4.23 0.59CR003336 31.31 5.47 30.48 5.10 0.83 0.75 CR003337 61.93 2.68 59.28 2.112.65 1.39 CR003338 68.00 6.09 65.40 6.78 2.60 1.17 CR003339 68.21 7.6712.37 1.47 55.84 6.31 CR003340 37.76 6.01 6.12 1.95 31.65 4.07 CR00334115.60 5.49 9.94 3.38 5.66 2.13 CR003342 11.06 6.71 10.78 6.69 0.28 0.03CR003343 45.41 15.20 40.05 10.79 5.36 5.20 CR003344 33.43 6.11 29.815.09 3.62 1.13 CR003345 10.58 9.25 6.12 5.38 4.45 3.87 CR003346 0.130.05 0.07 0.02 0.05 0.03 CR003347 22.57 10.94 21.08 11.19 1.49 0.90CR003348 38.44 10.45 17.04 5.04 21.40 5.89 CR003349 8.36 2.19 4.46 1.753.91 0.76 CR003350 29.60 5.17 25.16 4.56 4.44 0.67 CR003351 57.54 5.6731.98 2.63 25.57 3.08 CR003352 44.28 8.71 39.51 7.10 4.77 1.79 CR00335360.40 11.37 56.71 9.95 3.68 1.45 CR003354 5.36 3.94 4.84 3.41 0.53 0.71CR003355 15.80 5.38 12.36 4.23 3.44 1.16 CR003356 9.39 1.82 5.67 1.033.72 0.92 CR003357 45.83 10.66 42.37 8.47 3.46 2.28 CR003358 35.93 7.3428.66 7.76 7.27 1.77 CR003359 64.44 14.90 48.79 14.32 15.65 1.94CR003360 41.31 12.23 38.94 10.60 2.38 1.78 CR003361 14.05 4.79 11.474.35 2.58 0.43 CR003362 17.44 4.34 16.50 4.86 0.94 0.52 CR003363 42.659.90 28.58 6.95 14.07 3.01 CR003364 51.88 7.67 31.03 2.67 20.85 5.03CR003365 46.88 15.78 35.77 13.49 11.11 2.30 CR003366 54.69 9.10 46.208.98 8.49 1.11 CR003367 45.55 8.19 24.28 6.57 21.27 1.62 CR003368 51.558.60 48.34 9.87 3.22 1.36 CR003369 22.62 4.01 17.11 4.47 5.51 2.52CR003370 28.51 6.94 24.88 6.17 3.62 1.45 CR003371 15.91 4.17 14.07 4.021.84 0.22 CR003372 14.57 2.47 12.14 2.08 2.42 0.40 CR003373 17.69 8.4115.92 6.44 1.77 1.97 CR003374 5.43 0.53 5.12 0.62 0.31 0.36 CR0033752.06 0.04 1.96 0.06 0.10 0.03 CR003376 14.41 3.01 14.16 2.93 0.24 0.10CR003377 16.30 2.85 15.29 2.59 1.02 0.59 CR003378 8.16 3.83 6.82 3.431.34 0.61 CR003379 19.74 4.24 17.70 4.30 2.04 0.33 CR003380 17.08 2.4814.78 1.18 2.30 1.36 CR003381 6.81 3.48 6.18 3.82 0.63 0.44 CR0033821.73 0.14 1.58 0.12 0.15 0.03 CR003383 6.35 1.67 6.19 1.68 0.16 0.04CR003384 3.37 0.88 3.12 0.94 0.25 0.09 CR003385 53.94 9.41 46.32 10.667.62 1.29 CR003386 2.71 0.76 2.15 0.77 0.56 0.53 CR003387 1.39 0.15 1.270.17 0.12 0.02 CR003388 9.33 4.47 7.76 4.56 1.56 0.10 CR003389 31.846.09 27.27 5.96 4.57 1.21 CR003390 24.88 4.96 22.44 3.41 2.44 2.25CR003391 48.78 14.41 48.28 14.44 0.50 0.52 CR003392 14.64 5.25 14.324.95 0.33 0.36 CR005298 42.65 10.94 21.29 8.16 21.36 2.87 CR005299 38.615.57 36.32 3.99 2.30 2.11 CR005300 64.34 9.55 53.20 6.59 11.15 3.33CR005301 37.04 5.32 33.39 3.85 3.65 1.89 CR005302 33.21 2.19 30.93 2.432.29 0.24 CR005303 21.63 6.05 20.55 5.80 1.08 0.25 CR005304 62.82 3.288.07 1.22 54.75 4.27 CR005305 13.51 3.58 12.30 3.49 1.21 0.84 CR00530624.07 5.24 21.20 5.03 2.87 1.10 CR005307 22.03 3.86 7.70 1.35 14.33 4.15

Table 7 shows the average and standard deviation for % Edit, % Insertion(Ins), and % Deletion (Del) for the tested TTR dgRNAs electroporatedwith Spy Cas9 protein (RNP) in primary human hepatocytes.

TABLE 7 TTR editing data in primary human hepatocytes electroporatedwith Spy Cas9 protein loaded with dgRNAs Std Std Std Avg Dev Avg Dev AvgDev % % % % % % GUIDE ID Edit Edit Insert Insert Deletion DeletionCR003335 72.20 4.53 69.70 4.36 2.50 0.30 CR003336 39.17 3.04 38.43 3.200.70 0.17 CR003337 54.27 2.70 53.23 3.05 1.30 0.26 CR003338 83.03 4.8480.87 4.63 2.13 0.25 CR003339 43.00 2.66 8.93 1.86 34.07 1.72 CR00334012.03 1.55 5.60 1.32 6.50 0.53 CR003341 11.43 0.71 7.03 0.50 4.40 1.21CR003342 32.77 3.63 31.87 3.28 0.90 0.35 CR003343 77.10 2.21 75.63 2.011.50 0.36 CR003344 39.40 3.86 33.30 2.52 6.10 1.31 CR003345 48.07 6.2434.53 2.95 13.57 3.74 CR003346 35.67 1.80 20.83 1.65 14.83 1.66 CR00334782.30 5.93 81.97 5.98 0.43 0.15 CR003348 28.53 1.79 11.30 2.46 17.270.86 CR003349 4.10 0.17 2.33 0.46 1.87 0.25 CR003350 28.13 3.50 22.402.41 5.73 1.22 CR003351 51.77 5.11 30.83 3.32 20.97 2.43 CR003352 29.834.18 25.63 3.67 4.30 0.56 CR003353 84.83 4.68 82.23 4.05 2.63 0.74CR003354 2.50 0.36 2.43 0.32 0.03 0.06 CR003355 12.53 1.54 10.60 2.361.97 1.17 CR003356 9.97 2.68 7.80 2.01 2.23 0.85 CR003357 36.23 4.0235.47 4.11 0.77 0.61 CR003358 5.70 1.42 4.93 1.36 0.80 0.26 CR00335963.77 7.07 56.33 5.81 7.50 1.35 CR003360 32.23 3.09 31.67 2.97 0.63 0.31CR003361 4.10 0.36 3.73 0.42 0.37 0.06 CR003362 7.03 1.30 6.87 1.20 0.200.20 CR003363 9.43 8.22 7.80 6.86 1.63 1.44 CR003364 23.30 5.20 16.934.96 6.53 0.55 CR003365 42.37 3.88 35.57 1.88 6.83 2.00 CR003366 34.703.26 31.63 2.98 3.10 1.15 CR003367 39.20 5.31 22.93 4.14 16.37 1.46CR003368 28.47 3.29 27.63 2.90 0.80 0.66 CR003369 3.67 1.16 3.30 1.060.40 0.20 CR003370 15.27 1.75 14.43 1.72 0.90 0.20 CR003371 16.20 2.1314.47 2.37 1.87 0.81 CR003372 12.17 2.69 10.47 2.63 1.77 0.12 CR0033730.87 0.21 0.83 0.25 0.07 0.12 CR003374 0.80 0.17 0.70 0.26 0.10 0.10CR003375 1.33 1.10 1.27 1.08 0.07 0.06 CR003376 1.90 1.06 1.87 1.00 0.030.06 CR003377 10.23 1.53 10.13 1.51 0.10 0.10 CR003378 4.60 1.92 3.871.19 0.73 0.67 CR003379 6.57 1.00 6.30 0.70 0.27 0.31 CR003380 5.37 2.575.27 2.54 0.10 0.10 CR003381 6.20 2.74 5.83 2.61 0.50 0.10 CR003382 8.402.07 8.10 1.87 0.43 0.21 CR003383 8.57 0.75 3.37 0.67 5.27 0.46 CR0033841.87 0.67 1.73 0.57 0.23 0.12 CR003385 40.87 6.86 38.43 6.41 2.53 0.45CR003386 4.90 1.20 4.47 1.14 0.47 0.25 CR003387 1.87 0.25 1.70 0.26 0.200.10 CR003388 5.70 0.40 5.47 0.40 0.27 0.12 CR003389 27.67 2.76 27.202.88 0.50 0.36 CR003390 15.97 3.86 15.80 3.99 0.23 0.15 CR003391 29.773.85 29.57 3.85 0.27 0.06 CR003392 4.13 1.21 4.00 1.15 0.17 0.06CR005298 39.90 2.92 22.37 3.04 17.57 0.42 CR005299 8.65 0.78 8.30 0.990.35 0.21 CR005300 57.47 1.69 53.47 1.86 4.10 0.92 CR005301 25.37 1.6524.00 2.26 1.60 0.82 CR005302 61.10 5.20 60.10 4.77 1.00 0.46 CR00530353.57 8.52 53.07 8.36 0.53 0.47 CR005304 67.00 5.80 5.53 1.37 61.63 6.98CR005305 3.83 0.78 3.53 0.61 0.40 0.17 CR005306 9.43 1.63 8.07 2.17 1.370.72 CR005307 8.17 1.20 5.20 0.87 3.00 0.82

Table 8 shows the average and standard deviation for % Edit, % Insertion(Ins), and % Deletion (Del) for the tested TTR and control dgRNAstransfected with Spy Cas9 protein (RNP) in primary human hepatocytes.

TABLE 8 TTR editing data in primary human hepatocytes transfected withSpy Cas9 loaded with dgRNAs Std Std Std Avg Dev Avg Dev Avg Dev % % % %% % GUIDE ID Edit Edit Insert Insert Deletion Deletion CR001261 32.511.00 12.50 0.47 20.01 0.59 CR001262 50.09 1.48 45.25 1.69 4.83 0.31CR001263 15.25 2.41 14.83 2.37 0.42 0.10 CR001264 45.30 3.48 23.87 2.0921.43 1.68 CR003335 51.14 4.27 49.51 4.04 1.63 0.25 CR003336 30.70 2.4130.11 2.48 0.58 0.11 CR003337 49.43 4.75 47.54 4.49 1.88 0.47 CR00333861.34 3.55 59.13 3.44 2.22 0.11 CR003339 45.06 9.83 8.85 1.65 36.21 8.34CR003340 10.44 2.44 5.94 1.34 4.50 1.16 CR003341 19.66 3.67 14.64 3.315.02 0.37 CR003342 20.66 2.55 19.85 2.54 0.81 0.15 CR003343 43.25 4.4741.61 4.26 1.63 0.33 CR003344 35.45 13.12 30.97 11.72 4.48 1.51 CR00334528.90 6.33 21.00 5.23 7.91 1.81 CR003346 4.11 1.36 2.27 0.53 1.84 0.85CR003347 66.35 4.48 66.11 4.51 0.24 0.08 CR003348 23.18 2.16 13.74 1.179.44 0.99 CR003349 10.83 1.57 9.00 1.41 1.83 0.32 CR003350 24.84 2.7419.77 1.91 5.07 0.89 CR003351 40.28 1.31 23.92 0.70 16.36 0.78 CR00335230.48 1.93 27.27 2.31 3.21 0.38 CR003353 61.54 4.13 59.38 4.04 2.16 0.11CR003354 10.31 1.47 10.07 1.50 0.23 0.11 CR003355 19.11 0.92 17.69 0.791.42 0.44 CR003356 7.53 1.78 6.24 1.51 1.29 0.32 CR003357 49.35 2.5348.45 2.54 0.90 0.13 CR003358 31.62 5.97 25.95 5.03 5.67 1.04 CR00335959.47 6.05 50.96 5.69 8.51 0.54 CR003360 31.47 4.12 30.27 4.21 1.19 0.22CR003361 13.08 1.48 12.52 1.45 0.56 0.18 CR003362 11.65 1.24 11.10 1.060.56 0.36 CR003363 27.65 2.84 21.47 2.39 6.18 0.61 CR003364 35.29 3.5023.93 2.63 11.36 1.16 CR003365 47.78 3.67 40.24 3.12 7.54 0.72 CR00336642.74 3.41 37.95 2.88 4.79 0.60 CR003367 31.19 4.60 16.06 2.66 15.131.94 CR003368 34.83 5.05 33.83 5.09 1.00 0.10 CR003369 12.98 0.26 11.670.21 1.31 0.11 CR003370 20.06 1.79 18.80 1.65 1.26 0.28 CR003371 18.802.73 17.23 2.34 1.57 0.43 CR003372 17.56 2.26 15.74 2.16 1.81 0.10CR003373 3.64 0.29 3.44 0.30 0.19 0.07 CR003374 2.65 0.33 2.52 0.33 0.140.02 CR003375 5.04 0.66 4.93 0.66 0.11 0.01 CR003376 5.00 1.10 4.86 1.100.14 0.03 CR003377 12.77 2.00 12.45 1.84 0.31 0.18 CR003378 8.66 1.908.24 1.74 0.42 0.19 CR003379 16.86 2.62 16.51 2.62 0.34 0.08 CR0033808.17 1.42 7.71 1.47 0.46 0.10 CR003381 7.15 0.73 6.88 0.67 0.27 0.07CR003382 2.44 0.06 2.28 0.05 0.15 0.03 CR003383 4.76 0.40 4.52 0.42 0.240.09 CR003384 3.56 0.26 3.39 0.26 0.17 0.01 CR003385 41.15 6.06 38.155.59 3.00 0.48 CR003386 3.22 0.25 2.97 0.27 0.25 0.02 CR003387 1.79 0.111.68 0.09 0.11 0.04 CR003388 5.43 1.03 4.38 1.00 1.05 0.25 CR00338919.87 4.39 19.19 4.52 0.68 0.24 CR003390 16.09 2.84 15.85 2.91 0.24 0.09CR003391 34.72 8.29 34.46 8.35 0.26 0.06 CR003392 10.07 1.06 9.93 1.020.14 0.04 CR005298 32.07 1.02 21.12 1.02 10.95 0.15 CR005299 19.37 0.6118.79 0.51 0.58 0.13 CR005300 57.23 6.24 53.62 5.44 3.61 0.87 CR00530131.37 3.02 29.53 2.88 1.84 0.15 CR005302 48.29 5.22 47.32 5.32 0.97 0.14CR005303 36.45 4.83 36.06 4.72 0.39 0.12 CR005304 49.45 6.85 4.32 0.3145.13 6.74 CR005305 7.07 1.43 6.73 1.30 0.34 0.17 CR005306 18.81 1.8216.24 1.57 2.57 0.35 CR005307 18.73 1.68 10.18 0.92 8.55 0.88

Table 9 shows the average and standard deviation for % Edit, % Insertion(Ins), and % Deletion (Del) for the tested TTR and control dgRNAsco-transfected with Spy Cas9 mRNA (SEQ ID NO:2) in primary humanhepatocytes.

TABLE 9 TTR editing data in primary human hepatocytes transfected withSpy Cas9 mRNA and dgRNAs Std Std Std Avg Dev Avg Dev Avg Dev % % % % % %GUIDE ID Edit Edit Insert Insert Deletion Deletion CR001261 32.33 4.955.83 1.63 26.47 3.30 CR001262 41.50 4.71 34.43 3.31 7.13 1.42 CR00126310.23 3.61 9.40 3.20 0.90 0.44 CR001264 42.80 0.50 11.90 1.32 30.90 1.80CR003335 36.43 2.98 33.03 2.31 3.40 0.70 CR003336 16.93 3.78 16.20 3.410.80 0.44 CR003337 19.30 1.57 18.10 1.44 1.23 0.15 CR003338 36.30 9.5533.73 9.27 2.73 0.49 CR003339 36.43 1.21 2.27 0.15 34.23 1.31 CR00334024.97 2.78 1.83 0.23 23.17 2.66 CR003341 15.83 1.38 6.80 0.53 9.07 0.81CR003342 22.10 1.27 20.60 0.57 1.50 0.71 CR003343 55.03 0.38 52.40 0.532.60 0.44 CR003344 31.50 1.30 22.40 1.31 9.20 0.10 CR003345 50.65 2.9032.30 1.56 18.45 1.20 CR003346 19.97 1.94 5.63 0.55 14.33 1.72 CR00334741.47 3.59 41.33 3.63 0.17 0.06 CR003348 18.00 0.87 2.30 0.66 15.80 0.61CR003349 2.57 0.81 0.90 0.35 1.70 0.46 CR003350 26.63 4.25 16.33 2.4510.33 1.75 CR003351 26.50 1.61 10.20 0.92 16.37 0.97 CR003352 16.80 5.0311.73 3.86 5.07 1.14 CR003353 53.73 6.01 49.50 5.82 4.43 0.75 CR0033542.97 0.95 2.87 0.85 0.13 0.12 CR003355 12.07 2.61 10.47 2.08 1.63 0.59CR003356 7.27 0.72 4.70 0.53 2.67 0.21 CR003357 25.93 4.55 25.30 4.220.63 0.35 CR003358 3.90 0.79 2.73 0.45 1.17 0.51 CR003359 32.93 4.3425.67 3.25 7.33 1.24 CR003360 14.90 4.85 14.13 4.66 0.90 0.52 CR0033613.53 0.60 2.73 0.55 0.87 0.15 CR003362 6.60 1.47 6.17 1.45 0.47 0.21CR003363 16.70 1.08 11.80 0.79 4.93 0.60 CR003364 15.63 2.45 6.73 0.818.93 1.70 CR003365 26.90 3.05 20.23 2.02 6.67 1.16 CR003366 24.53 1.2620.47 1.45 4.07 0.23 CR003367 37.33 1.40 14.03 0.40 23.37 1.25 CR00336811.10 1.91 10.53 1.90 0.60 0.10 CR003369 1.60 0.46 0.90 0.20 0.70 0.36CR003370 2.83 0.57 2.33 0.40 0.50 0.17 CR003371 3.40 0.80 2.67 0.75 0.730.15 CR003372 1.77 0.75 1.13 0.57 0.63 0.23 CR003373 1.40 0.36 1.00 0.350.37 0.12 CR003374 0.27 0.21 0.27 0.21 0.03 0.06 CR003375 1.27 0.64 1.230.58 0.03 0.06 CR003376 2.83 0.81 2.73 0.81 0.13 0.06 CR003377 17.536.35 16.97 6.11 0.57 0.25 CR003378 9.80 1.37 8.50 1.21 1.37 0.15CR003379 13.20 1.18 12.00 1.05 1.27 0.15 CR003380 2.93 0.58 2.47 0.570.47 0.15 CR003381 4.07 1.21 3.33 0.96 0.73 0.25 CR003382 0.97 0.25 0.970.25 0.00 0.00 CR003383 15.70 3.22 2.07 0.35 13.70 2.82 CR003384 1.700.62 1.50 0.56 0.20 0.10 CR003385 36.77 0.70 33.23 0.74 3.60 0.26CR003386 8.27 1.63 8.20 1.57 0.13 0.06 CR003387 7.87 1.58 7.80 1.64 0.030.06 CR003388 12.97 1.30 11.87 1.21 1.17 0.25 CR003389 44.27 1.72 41.471.59 2.83 0.15 CR003390 20.23 2.08 18.73 1.92 1.60 0.17 CR003391 15.475.87 15.20 5.72 0.30 0.10 CR003392 2.43 0.55 2.37 0.59 0.07 0.06CR005298 15.70 2.79 4.13 0.87 11.60 2.00 CR005299 9.43 0.68 8.93 0.680.60 0.00 CR005300 31.53 3.44 27.60 2.77 3.97 0.76 CR005301 6.77 1.445.47 0.96 1.40 0.61 CR005302 34.80 7.17 33.67 7.01 1.13 0.21 CR00530335.50 5.90 35.00 5.81 0.50 0.10 CR005304 45.27 4.71 0.83 0.15 44.47 4.57CR005305 7.53 1.06 5.93 1.10 1.60 0.10 CR005306 9.97 0.38 7.13 0.23 2.870.12 CR005307 12.90 2.43 3.67 0.61 9.30 1.80

Table 10 shows the average and standard deviation for % Edit, %Insertion (Ins), and % Deletion (Del) for the tested TTR dgRNAselectroporated with Spy Cas9 protein (RNP) in primary cyno hepatocytes.

TABLE 10 TTR editing data in primary cyno hepatocytes electroporatedwith Spy Cas9 protein and dgRNAs Std Std Std Avg Dev Avg Dev Avg Dev % %% % % % GUIDE ID Edit Edit Insert Insert Deletion Deletion CR003336 8.181.93 8.10 1.94 0.07 0.01 CR003337 24.94 5.80 24.10 4.71 0.84 1.10CR003338 44.94 9.99 44.89 9.97 0.05 0.01 CR003339 8.95 0.89 4.93 0.644.02 0.25 CR003340 12.53 2.22 7.72 0.13 4.80 2.09 CR003341 8.43 10.537.66 9.91 0.77 0.63 CR003344 35.72 4.67 33.81 5.29 1.91 0.61 CR00334552.92 3.26 30.74 0.78 22.19 2.48 CR003346 1.91 0.86 1.82 0.82 0.09 0.04CR003347 72.41 0.38 72.15 0.73 0.25 0.34 CR003352 1.25 0.20 1.16 0.210.09 0.01 CR003353 4.75 0.43 4.67 0.47 0.08 0.04 CR003358 20.47 0.3019.01 0.51 1.46 0.21 CR003359 46.17 1.14 40.66 2.00 5.51 0.86 CR00336029.47 0.63 29.05 1.00 0.42 0.37 CR003361 4.53 0.14 4.46 0.18 0.08 0.04CR003362 4.59 0.80 4.36 0.77 0.22 0.03 CR003363 15.64 1.92 13.24 2.652.39 0.73 CR003364 19.62 2.54 14.27 2.72 5.35 0.17 CR003365 10.31 1.819.33 1.80 0.97 0.01 CR003366 18.52 0.71 17.62 0.33 0.90 0.39 CR00336818.56 3.89 18.30 3.77 0.26 0.11 CR003369 1.53 0.25 1.28 0.40 0.25 0.15CR003370 2.52 0.64 2.40 0.63 0.12 0.01 CR003371 1.83 0.38 1.69 0.41 0.140.03 CR003372 2.15 0.30 1.83 0.33 0.32 0.04 CR003382 10.86 2.04 8.541.93 2.33 0.11 CR003383 8.86 2.30 4.31 0.69 4.55 1.61 CR003384 3.75 0.352.50 0.37 1.25 0.02 CR003385 30.96 1.61 26.84 2.20 4.12 0.59 CR0033865.54 1.42 3.51 1.26 2.03 0.15 CR003387 4.72 0.03 4.55 0.08 0.17 0.11CR003388 6.81 0.17 6.59 0.28 0.22 0.11 CR003389 18.83 4.99 18.05 4.920.78 0.07 CR003390 16.87 3.88 16.49 3.48 0.39 0.39 CR003391 36.44 1.0935.73 1.37 0.71 0.28 CR003392 7.02 0.97 6.63 0.59 0.38 0.37 CR00529913.48 2.96 13.23 2.74 0.26 0.22 CR005301 46.76 1.75 46.34 2.19 0.42 0.44CR005302 1.34 0.19 1.26 0.19 0.08 0.00 CR005303 59.28 1.05 58.72 1.060.56 0.00 CR005305 11.28 0.39 11.13 0.39 0.15 0.00 CR005307 4.56 0.712.01 0.49 2.55 0.21

Table 11 shows the average and standard deviation for % Edit, %Insertion (Ins), and % Deletion (Del) for the tested cyno specific TTRdgRNAs electroporated with Spy Cas9 protein (RNP) on primary cynohepatocytes.

TABLE 11 TTR editing data in primary cyno hepatocytes electroporatedwith Spy Cas9 protein and cyno specific dgRNAs Std Std Std Avg Dev AvgDev Avg Dev % % % % % % GUIDE ID Edit Edit Insert Insert DeletionDeletion CR000689 24.41 1.67 18.11 2.41 6.30 0.93 CR005364 27.70 0.740.58 0.29 27.11 0.60 CR005365 64.94 2.03 0.10 0.04 64.85 2.05 CR00536677.00 1.17 0.33 0.27 76.67 0.99 CR005367 50.79 0.53 0.53 0.25 50.26 0.36CR005368 27.60 2.07 0.33 0.45 27.27 2.32 CR005369 42.01 0.33 8.09 0.5533.92 0.31 CR005370 63.52 3.21 0.59 0.33 62.93 2.88 CR005371 8.42 0.690.31 0.12 8.10 0.57 CR005372 17.98 1.39 0.83 0.77 17.16 0.71

Example 3. Screening of sgRNA Sequences

Cross Screening of TTR sgRNAs in Multiple Cell Types

Guides in modified sgRNA format targeting human and/or cyno TTR weredelivered to primary human hepatocytes and primary cyno hepatocytes asdescribed in Example 1. Percent editing was determined for crRNAscomprising each guide sequence across each cell type and the guidesequences were then rank ordered based on highest % edit. The screeningdata for the guide sequences in Table 2 in both cell lines are listedbelow (Table 12 through 15).

Table 12 shows the average and standard deviation for % Edit, %Insertion (Ins), and % Deletion (Del) for the tested TTR sgRNAstransfected with Spy Cas9 protein (RNP) in primary human hepatocytes.

TABLE 12 TTR editing data in primary human hepatocytes transfected withSpy Cas9 protein and sgRNAs Std Std Std Avg Dev Avg Dev Avg Dev % % % %% % GUIDE ID Edit Edit Insert Insert Deletion Deletion G000480 81.801.98 77.15 2.19 4.70 0.28 G000481 46.90 1.71 27.77 3.88 19.43 4.76G000482 66.67 2.35 56.57 4.14 10.10 1.85 G000483 47.90 6.56 19.57 3.3728.50 3.25 G000484 62.97 0.90 29.23 0.21 33.83 0.95 G000485 56.07 3.3753.07 2.84 3.13 0.60 G000486 69.73 6.86 9.83 1.93 59.93 5.63 G00048767.30 2.75 65.27 3.41 2.07 1.06 G000488 61.27 1.95 26.30 1.55 35.00 1.30G000489 60.17 2.75 51.07 3.18 9.43 0.45 G000490 55.90 7.88 46.13 7.559.80 0.69 G000491 74.30 1.55 70.27 2.37 4.33 0.72 G000492 60.97 5.8157.90 4.64 3.13 1.35 G000493 41.40 3.08 38.90 3.29 2.67 0.35 G00049462.23 3.30 61.47 3.25 0.77 0.31 G000495 50.80 1.85 45.80 1.25 5.37 0.64G000496 72.33 1.63 44.73 2.14 27.67 1.46 G000497 59.67 1.40 51.10 1.148.73 0.71 G000498 72.80 3.75 60.17 3.12 12.70 0.72 G000499 66.40 3.5565.23 3.72 1.17 0.38 G000500 65.53 1.21 62.00 1.11 3.83 0.40 G00050160.93 1.91 55.13 1.43 6.00 0.56

Table 13 shows the average and standard deviation at 12.5 nM for % Edit,% Insertion (Ins), and % Deletion (Del) for the tested TTR sgRNAsco-transfected with Spy Cas9 mRNA (SEQ ID NO:2) in primary humanhepatocytes.

TABLE 13 TTR editing data in primary human hepatocytes transfected withSpy Cas9 mRNA and sgRNAs Std Std Std Avg Dev Avg Dev Avg Dev % % % % % %GUIDE ID Edit Edit Insert Insert Deletion Deletion G000480 73.28 0.6159.85 0.13 13.47 0.51 G000481 34.30 5.26 14.62 2.59 19.77 2.72 G00048240.93 3.95 27.70 2.92 13.25 0.97 G000483 27.82 2.93 4.05 0.51 23.85 2.43G000484 43.37 6.79 13.98 2.61 29.48 4.15 G000485 30.82 5.76 28.87 5.501.97 0.28 G000486 59.13 5.62 2.82 0.86 56.37 4.92 G000487 49.57 0.9947.38 0.89 2.27 0.24 G000488 49.40 5.05 11.98 1.40 37.48 3.68 G00048924.25 2.82 14.17 2.01 10.28 1.38 G000490 24.72 2.35 19.38 2.04 5.38 0.41G000491 45.93 1.22 42.42 1.06 3.60 0.33 G000492 34.65 2.21 32.45 2.012.22 0.25 G000493 11.55 1.35 10.65 1.58 0.97 0.30 G000494 26.22 4.0325.17 3.89 1.07 0.15 G000495 47.77 1.88 43.40 1.91 4.45 0.17 G00049663.30 2.60 11.08 2.10 52.25 0.67 G000497 40.33 3.32 34.48 2.71 5.85 0.61G000498 60.02 5.42 45.20 4.34 14.90 1.08 G000499 39.30 6.04 38.58 5.860.77 0.12 G000500 35.50 0.61 32.47 0.49 3.10 0.18 G000501 40.32 1.5033.82 2.04 6.62 0.55 G000567 27.28 7.59 17.35 4.72 10.02 2.94 G00056843.75 5.83 43.00 5.81 0.80 0.18 G000570 68.42 3.64 68.08 3.61 0.35 0.00G000571 20.47 3.41 14.47 2.72 6.13 0.78 G000572 55.42 8.13 41.62 6.4813.85 1.60

Table 14 shows the average and standard deviation for % Edit, %Insertion (Ins), and % Deletion (Del) for the tested TTR sgRNAselectroporated with Spy Cas9 protein (RNP) on primary cyno hepatocytes.Note that guides G000480 and G000488 have one mismatch to cyno, whichmay compromise their editing efficiency in cyno cells.

TABLE 14 TTR editing data in primary cyno hepatocytes electroporatedwith Spy Cas9 protein and sgRNAs Std Std Std Avg Dev Avg Dev Avg Dev % %% % % % GUIDE ID Edit Edit Insert Insert Deletion Deletion G000480 10.200.56 9.83 0.81 0.37 0.25 G000481 69.13 8.62 33.73 2.67 35.50 11.23G000482 75.17 2.34 55.23 2.00 20.03 0.85 G000485 22.93 0.95 22.00 0.821.07 0.21 G000486 79.90 0.79 11.90 0.85 68.07 0.35 G000488 9.63 0.505.37 0.38 4.27 0.35 G000489 67.53 1.15 53.53 1.56 14.17 0.64 G00049061.67 0.72 54.47 1.10 7.27 1.23 G000491 66.20 1.11 64.37 0.47 1.90 0.70G000493 50.13 0.74 48.07 1.69 2.10 0.98 G000494 81.53 0.71 79.57 0.492.07 0.67 G000498 91.37 1.48 68.50 1.64 22.87 1.50 G000499 83.40 0.3682.00 0.20 1.43 0.55 G000500 45.20 3.66 42.60 3.80 2.63 0.25

Table 15 shows the average and standard deviation for % Edit, %Insertion (Ins), and % Deletion (Del) for the tested cyno specific TTRsgRNAs electroporated with Spy Cas9 protein (RNP) on primary cynohepatocytes.

TABLE 15 TTR editing data in primary cyno hepatocytes electroporatedwith Spy Cas9 protein and cyno specific sgRNAs (e.g., those having ananalogous human gRNA, See Table 3) Std Std Std Avg Dev Avg Dev Avg Dev %% % % % % GUIDE ID Edit Edit Insert Insert Deletion Deletion G00050295.10 0.96 13.97 1.69 81.27 2.60 G000503 58.53 2.40 52.07 1.68 6.50 2.46G000504 77.17 0.96 69.73 1.29 7.53 0.57 G000505 95.53 1.06 95.50 1.010.10 0.10 G000506 89.43 1.36 86.90 1.64 3.07 0.42 G000507 71.17 3.2267.03 2.39 4.60 1.65 G000508 45.63 3.01 41.57 2.95 4.17 0.91 G00050993.03 0.81 43.60 1.30 49.73 1.76 G000510 90.80 0.53 89.13 0.40 1.77 0.12G000511 62.77 1.63 60.87 1.55 2.00 0.35

Example 4. Screening of Lipid Nanoparticle (LNP) Formulations ContainingSpy Ca9 mRNA and sgRNA

Cross screening of LNP formulated TTR sgRNAs with Spy Cas9 mRNA inprimary human hepatocytes and primary cyno hepatocytes.

Lipid nanoparticle formulations of modified sgRNAs targeting human TTRand the cyno matched sgRNA sequences were tested on primary humanhepatocytes and primary cyno hepatocytes in a dose response curve.Primary human and cyno hepatocytes were plated as described inExample 1. Both cell lines were incubated at 37° C., 5% CO₂ for 24 hoursprior to treatment with LNPs. The LNPs used in the experiments detailedin Tables 16-19 were prepared using the Nanoassemblr™ procedure, eachcontaining the specified sgRNA and Cas9 mRNA (SEQ ID NO:2), each havingLipid. The LNPs contained Lipid A, Cholesterol, DSPC, and PEG2k-DMG in a45:44:9:2 molar ratio, respectively, and had a N:P ratio of 4.5. LNPswere incubated in hepatocyte maintenance media containing 6% cyno serumat 37° C. for 5 minutes. Post incubation the LNPs were added onto theprimary human or cyno hepatocytes in an 8 point 2-fold dose responsecurve starting at 100 ng mRNA. The cells were lysed 72 hours posttreatment for NGS analysis as described in Example 1. Percent editingwas determined for crRNAs comprising each guide sequence across eachcell type and the guide sequences were then rank ordered based onhighest % editing at 12.5 ng mRNA input and 3.9 nM guide concentration.The dose response curve data for the guide sequences in both cell linesis shown in FIGS. 4 through 7. The % editing at 12.5 ng mRNA input and3.9 nM guide concentration are listed below (Table 16 through 18).

Table 16 shows the average and standard deviation at 12.5 ng of cas9mRNA for % Edit, % Insertion (Ins), and % Deletion (Del) for the testedTTR sgRNAs formulated in lipid nanoparticles with Spy Cas9 mRNA onprimary human hepatocytes as dose response curves. G000570 exhibited anuncharacteristic dose response curve compared to the other sgRNAs whichmay be an artifact of the experiment. The data are shown graphically inFIG. 4.

TABLE 16 TTR editing data in primary human hepatocytes treated with LNPformulated Spy Cas9 mRNA (SEQ ID NO: 2) and sgRNAs 12.5 ng mRNA, 3.9 nMsgRNA GUIDE ID Avg % Edit Std Dev % Edit G000480 59.33 0.73 G00048124.37 0.37 G000482 19.10 2.64 G000483 7.37 0.67 G000484 16.67 1.23G000485 14.23 2.36 G000486 61.33 2.59 G000487 17.37 0.95 G000488 44.803.00 G000489 16.85 0.06 G000490 10.53 1.90 G000491 31.60 2.33 G00049215.87 0.44 G000493 7.33 0.73 G000494 6.37 1.07 G000495 23.97 1.66G000496 30.73 3.76 G000497 15.10 3.30 G000498 24.43 1.30 G000499 16.071.67 G000500 23.57 2.44 G000501 32.30 2.49 G000567 48.95 1.06 G00056854.60 3.68 G000570 88.30 1.84 G000572 55.45 1.20

Table 17 shows the average and standard deviation at 12.5 ng of mNRA and3.9 nM guide concentration for % Edit, % Insertion (Ins), and % Deletion(Del) for the tested TTR sgRNAs formulated in lipid nanoparticles withSpy Cas9 mRNA on primary cyno hepatocytes as dose response curves. Thedata are shown graphically in FIG. 5.

TABLE 17 TTR editing data in primary cyno hepatocytes treated with LNPformulated Spy Cas9 mRNA (SEQ ID NO: 2) and sgRNAs 12.5 ng mRNA, 3.9 nMsgRNA, GUIDE ID Avg % Edit Std Dev % Edit G000480 0.73 0.15 G00048149.20 1.39 G000482 26.13 5.33 G000483 0.73 0.60 G000484 0.10 0.00G000485 1.43 1.02 G000489 31.87 2.40 G000490 15.23 1.08 G000491 6.370.38 G000492 0.70 0.28 G000493 7.63 1.14 G000494 14.30 1.06 G000495 0.730.06 G000497 0.23 0.06 G000498 37.90 1.42 G000499 14.63 0.70 G00050010.47 0.32 G000501 1.37 0.31 G000567 0.10 0.00 G000568 9.25 0.21 G00057017.30 0.85 G000571 20.20 2.26 G000572 30.60 0.42

Table 18 shows the average and standard deviation at 12.5 ng of mRNA and3.9 nM guide concentration for % Edit, % Insertion (Ins), and % Deletion(Del) for the tested cyno specific TTR sgRNAs formulated in lipidnanoparticles with Spy Cas9 mRNA on primary cyno hepatocytes as doseresponse curves. The data are shown graphically in FIG. 6.

TABLE 18 TTR editing data in primary cyno hepatocytes treated with LNPformulated Spy Cas9 mRNA (SEQ ID NO: 2) and cyno matched sgRNAs 12.5 ngmRNA, 3.9 nM sgRNA GUIDE ID % Edit Std Dev % Edit G000502 80.70 0.14G000506 60.13 0.70 G000509 74.47 7.28 G000510 61.87 2.54Cross Screening of LNP Formulated TTR sgRNAs with Spy Cas9 mRNA inPrimary Human Hepatocytes and Primary Cyno Hepatocytes

Lipid nanoparticle formulations of modified sgRNAs targeting human TTRand the cyno matched sgRNA sequences were tested on primary humanhepatocytes and primary cyno hepatocytes in a dose response curve.Primary human and cyno hepatocytes were plated as described inExample 1. Both cell lines were incubated at 37° C., 5% CO₂ for 24 hoursprior to treatment with LNPs. The LNPs used in the experiments detailedin Tables 20-22 were prepared using the cross-flow procedure describedabove but purified using PD-10 columns (GE Healthcare Life Sciences) andconcentrated using Amicon centrifugal filter units (Millipore Sigma),each containing the specified sgRNA and Cas9 mRNA (SEQ ID NO:1). TheLNPs contained Lipid A, Cholesterol, DSPC, and PEG2k-DMG in a 50:38:9:3molar ratio, respectively, and had a N:P ratio of 6.0. LNPs wereincubated in hepatocyte maintenance media containing 6% cyno serum at37° C., 5% CO₂ for 5 minutes. Post incubation the LNPs were added ontothe primary human or cyno hepatocytes in an 8 point 3-fold dose responsecurve starting at 300 ng mRNA. The cells were lysed 72 hours posttreatment for NGS analysis as described in Example 1. Percent editingwas determined for crRNAs comprising each guide sequence across eachcell type and the guide sequences were then rank ordered based on EC50values and maximum editing percent. The dose response curve data for theguide sequences in both cell lines is shown in FIGS. 4 through 7. The EC50 values and maximum editing percent are listed below (Table 19 through22).

Table 19 shows the EC50 and maximum editing the tested human specificTTR sgRNAs formulated in lipid nanoparticles with U-depleted Spy Cas9mRNA on primary human hepatocytes as dose response curves. The data areshown graphically in FIG. 4.

TABLE 19 TTR editing data in primary human hepatocytes treated with LNPformulated Spy Cas9 mRNA and human specific sgRNAs GUIDE ID EC50 MaxEditing G000480 0.10 98.69 G000481 1.43 87.05 G000482 0.65 97.02 G0004831.88 77.39 G000484 0.95 94.14 G000488 0.72 95.83 G000489 1.38 86.33G000490 1.52 94.16 G000493 2.42 63.95 G000494 1.28 75.70 G000499 0.6396.31 G000500 0.39 88.70 G000568 0.78 95.72 G000570 0.23 98.22 G0005712.21 71.28 G000572 0.42 97.94

Table 20 shows the EC50 and maximum editing the tested human specificTTR sgRNAs formulated in lipid nanoparticles with U-depleted Spy Cas9mRNA on primary cyno hepatocytes as dose response curves. The data areshown graphically in FIG. 16.

TABLE 20 TTR editing data in primary cyno hepatocytes treated with LNPformulated Spy Cas9 mRNA and human specific sgRNAs GUIDE ID EC50 MaxEditing G000480 5.28 20.32 G000481 0.93 95.07 G000482 0.89 97.47 G0004834.40 56.52 G000484 3.47 0.22 G000488 11.56 21.63 G000489 1.79 89.21G000490 3.09 90.76 G000493 4.97 61.15 G000494 2.77 60.84 G000499 2.0074.94 G000500 4.42 58.04 G000567 1.76 97.06 G000568 1.87 87.93 G0005702.00 96.73 G000571 1.55 97.03 G000572 0.79 100.31

Table 21 shows the EC50 and maximum editing the tested cyno matched TTRsgRNAs formulated in lipid nanoparticles with U-depleted Spy Cas9 mRNAon primary human hepatocytes as dose response curves. The data are showngraphically in FIG. 17.

TABLE 21 TTR editing data in primary human hepatocytes treated with LNPformulated Spy Cas9 mRNA and cyno specific sgRNAs GUIDE ID EC50 MaxEditing G000502 0.70 91.50 G000504 5.16 7.16 G000505 3.57 13.48 G0005061.26 89.49

Table 22 shows the EC50 and maximum editing the tested cyno matched TTRsgRNAs formulated in lipid nanoparticles with U-depleted Spy Cas9 mRNAon primary cyno hepatocytes as dose response curves. The data are showngraphically in FIG. 18.

TABLE 22 TTR editing data in primary cyno hepatocytes treated with LNPformulated Spy Cas9 mRNA and cyno specific sgRNAs GUIDE ID EC50 MaxEditing G000502 0.26 100.05 G000503 2.26 83.41 G000504 1.42 98.04G000505 1.10 99.97 G000506 0.66 99.18

Example 5. Off-Target Analysis of TTR dgRNAs and sgRNAs Off-TargetAnalysis of TTR Guides

An oligo insertion based assay (See, e.g., Tsai et al., NatureBiotechnology 33, 187-197; 2015) was used to determine potentialoff-target genomic sites cleaved by Cas9 targeting TTR. Forty-fivedgRNAs from Table 1 (and two control guides with known off-targetprofiles) were screened in the HEK293_Cas9 cells. The human embryonickidney adenocarcinoma cell line HEK293 constitutively expressing SpyCas9 (“HEK293_Cas9”) was cultured in DMEM media supplemented with 10%fetal bovine serum and 500 μg/ml G418. Cells were plated at a density of30,000 cells/well in a 96-well plate 24 hours prior to transfection.Cells were transfected with Lipofectamine RNAiMAX (ThermoFisher, Cat.13778150) per the manufacturer's protocol. Cells were transfected with alipoplex containing individual crRNA (15 nM), trRNA (15 nM), and donoroligo with (10 nM) Lipofectamine RNAiMAX (0.3 μL/well) and OptiMem.Cells were lysed 24 hours post transfection and genomic DNA wasextracting using Zymo's Quick gDNA 96 Extraction kit (catalog # D3012)following the manufacturer's recommended protocol. The gDNA wasquantified using the Qubit High Sensitivity dsDNA kit (LifeTechnologies). Libraries were prepared per the previously describedmethod in Tsai et al, 2015 with minor modifications. Sequencing wasperformed on Illumina's MiSeq and HiSeq 2500. The assay identifiedpotential off-target sites for some of the crRNAs which are plotted inFIG. 2.

Table 23 shows the number of off-target integration sites detected inHekCas9 cells transfected with TTR dgRNAs along with a double strandedDNA oligo donor sequence.

TABLE 23 Number of off-target integration sites detected for TTR dgRNAsvia an oligo insertion based assay GUIDE ID # Sites CR003335 0 CR0033362 CR003337 10 CR003338 2 CR003339 3 CR003340 0 CR003342 0 CR003343 2CR003344 0 CR003345 0 CR003346 0 CR003347 1 CR003348 3 CR003351 1CR003352 2 CR003353 2 CR003355 1 CR003356 4 CR003357 3 CR003359 6CR003360 0 CR003363 4 CR003365 3 CR003366 1 CR003367 1 CR003368 2CR003369 2 CR003377 0 CR003380 0 CR003382 34 CR003383 1 CR003385 3CR003386 1 CR003387 6 CR003388 2 CR003389 2 CR003390 1 CR003391 0CR003392 0 CR005298 0 CR005300 0 CR005301 0 CR005302 1 CR005303 1CR005304 0

Additionally, a subset of the guides was assessed for off-targetpotential as modified sgRNAs in the Hek_Cas9 cells via the oligo basedinsertion method described above. The off-target results were plotted inFIG. 4.

Table 24 shows the number of off-target integration sites detected inHekCas9 cells transfected with TTR sgRNAs along with a double strandedDNA oligo donor sequence.

TABLE 24 Number of off-target integration sites detected for TTR sgRNAsvia an insertion detection method GUIDE ID # Sites G000480 11 G000481 3G000482 13 G000483 5 G000484 7 G000485 22 G000486 12 G000487 14 G0004880 G000489 19 G000490 12 G000491 28 G000492 97 G000493 7 G000494 4G000495 13 G000496 1 G000497 26 G000498 82 G000499 4 G000500 46 G0005014 G000567 9 G000568 937 G000570 19 G000571 16 G000572 15

Example 6. Targeted Sequencing for Validating Potential Off-Target Sites

The HEK293_Cas9 cells used in Example 5 for detecting potentialoff-targets constitutively overexpress Cas9, leading to a higher numberof potential off-target “hits” as compared to a transient deliveryparadigm in various cell types. Further, when delivering sgRNAs (asopposed to dgRNAs), the number of potential off-target hits may befurther inflated as sgRNA molecules are more stable than dgRNAs(especially when chemically modified). Accordingly, potential off-targetsites identified by an oligo insertion method as used in Example 5 maybe validated using targeted sequencing of the identified potentialoff-target sites.

In one approach, primary hepatocytes are treated with LNPs comprisingCas9 mRNA and a sgRNA of interest (e.g., a sgRNA having potentialoff-target sites for evaluation). The primary hepatocytes are then lysedand primers flanking the potential off-target site(s) are used togenerate an amplicon for NGS analysis. Identification of indels at acertain level may validate potential off-target site, whereas the lackof indels found at the potential off-target site may indicate a falsepositive in the HEK293_Cas9 cell assay.

Example 7. Phenotypic Analysis Western Blot Analysis of Secreted TTR

The hepatocellular carcinoma cell line, HepG2, was transfected asdescribed in Example 1 with select guides from Table 1 in triplicate.Two days post-transfection, one replicate was harvested for genomic DNAand analysis by NGS sequencing for editing efficiency. Five dayspost-transfection, media without serum was replaced on one replicate.After 4 hrs the media was harvested for analysis of secreted TTR by WBas previously described. The data for % edit for each guide andreduction of extracellular TTR is provided in FIG. 7.

Western Blot Analysis of Intracellular TTR

The hepatocellular carcinoma cell line, HUH7, was transfected asdescribed in Example 1 with crRNA comprising the guides from Table 1.The transfected pools of cells were retained in tissue culture andpassaged for further analysis. At seven days post-transfection, cellswere harvested and whole cell extracts (WCEs) were prepared andsubjected to analysis by Western Blot as previously described.

WCEs were analyzed by Western Blot for reduction of TTR protein. Fulllength TTR protein has a predicted molecular weight of −16 kD. A band atthis molecular weight was observed in the control lanes in the WesternBlot.

Percent reduction of TTR protein was calculated using the Licor OdysseyImage Studio Ver 5.2 software. GAPDH was used as a loading control andprobed simultaneously with TTR. A ratio was calculated for thedensitometry values for GAPDH within each sample compared to the totalregion encompassing the TTR band. Percent reduction of TTR protein wasdetermined after the ratios were normalized to control lanes. Resultsare shown in FIG. 8.

Example 8. LNP Delivery to Humanized TTR Mice and Mice Having Wt(Murine) TTR

Mice humanized with respect to the TTR gene were dosed with LNPformulations 701-704 containing the guides indicated in Table 25 (5 miceper formulation). These humanized TTR mice were engineered such that aregion of the endogenous murine TTR locus was deleted and replaced withan orthologous human TTR sequence so that the locus encodes a human TTRprotein. For comparison, 6 mice with murine TTR were dosed with LNP700,containing a guide (G000282) targeting murine TTR. LNPs with FormulationNumbers 1-5 in Table 25 were prepared using the Nanoassemblr™ procedureas described above while LNPs with Formulation Numbers 6-16 wereprepared using the cross-flow procedure described above but purifiedusing PD-10 columns (GE Healthcare Life Sciences) and concentrated usingAmicon centrifugal filter units (Millipore Sigma). As negative controls,mice of the corresponding genotype were dosed with vehicle alone(Tris-saline-sucrose buffer (TSS)). The background of the humanized TTRmice administered LNPs with Formulation Numbers 2-5 in Table 25 was 50%12956/SvEvTac 50% C57BL/6NTac; the background of the humanized TTR miceadministered LNPs having Formulation Numbers 6-16 in Table 25 as well asthe mice with murine TTR (administered LNP700, Formulation Number 1) was75% C57BL/6NTac 25% 12956/SvEvTac.

TABLE 25 LNP formulations for dosing humanized TTR mice. Molar Ratios(Lipid A, RNA Cholesterol, concen- DSPC, and Formulation tration N:PPEG2k-DMG, Number LNP Guide (mg/ml) Ratio respectively) 1 LNP700 G0002820.53 4.5 45:44:9:2 2 LNP701 G000481 0.46 4.5 45:44:9:2 3 LNP702 G0004890.61 4.5 45:44:9:2 4 LNP703 G000494 0.57 4.5 45:44:9:2 5 LNP704 G0004990.59 4.5 45:44:9:2 6 LNP1148 G000481 0.73 4.5 45:44:9:2 7 LNP1152G000499 0.45 6.0 50:38:9:3 8 LNP1153 G000482 0.53 6.0 50:38:9:3 9LNP1155 G000571 0.70 6.0 50:38:9:3 10 LNP1156 G000572 0.58 6.0 50:38:9:311 LNP1157 G000480 0.84 6.0 50:38:9:3 12 LNP1159 G000488 0.79 6.050:38:9:3 13 LNP1160 G000493 0.71 6.0 50:38:9:3 14 LNP1161 G000500 0.666.0 50:38:9:3 15 LNP1162 G000567 0.69 6.0 50:38:9:3 16 LNP1163 G0005700.66 6.0 50:38:9:3

LNPs having Formulation numbers 1-5 contained Cas9 mRNA of SEQ ID NO:2and LNPs having Formulation Numbers 6-16 contained Cas9 mRNA of SEQ IDNO: 1, all in a 1:1 ratio by weight to the guide. The LNPs containedLipid A, Cholesterol, DSPC, and PEG2k-DMG in the molar ratios recited inTable 25, respectively. Dosing with LNPs having Formulation Numbers 1-5was at 2 mg/kg (total RNA content) and dosing with LNPs havingFormulation Numbers 6-16 was at 1 mg/kg (total RNA content). Liverediting results were determined using primers designed to amplify theregion of interest for NGS analysis. Liver editing results forFormulation Numbers 1-5 are shown in FIG. 9 and indicate editing of thehuman TTR sequence with each of the four guides tested at a level >35%editing (mean values) with G000494 and G000499 providing values near60%. Liver editing results for formulation numbers 6-8, 10-13, and 15-16are shown in FIG. 13 and Table 26, which show efficient editing of thehuman TTR sequence with each of the formulations tested. Greater than38% editing was seen for all formulations, with several formulationsproviding editing values greater than 60%. Formulations 9 and 14 are notshown due to the design of the PCR amplicon and a resulting low numberof sequencing reads.

The level of human TTR in serum was measured in the mice providedformulation numbers 6-8, 10-13, and 15-16. See FIG. 14B. FIG. 14A is arepeat of FIG. 13 provided for comparison purposes. Knockdown of serumhuman TTR was detected for each formulation tested, which correlatedwith the amount of editing detected in liver (See FIG. 14A vs 14B, Table26).

TABLE 26 GUIDE ID % Editing Serum TTR(% TSS) TSS (vehicle) 0.06 100 G48161.28 10.52 G499 65.66 8.39 G482 70.86 4.65 G572 73.52 2.11 G480 77.343.48 G488 59.125 27.78 G493 38.55 49.73 G567 47.525 44.24 G570 45.541.73 G571 33.88 11.39 G500 44.44 34.28

In another set of experiments, humanized TTR mice were dosed with LNPformulations across a range of doses with guides G000480, G000488,G000489 and G000502. The formulations contained Cas9 mRNA (SEQ ID NO: 1)in a 1:1 ratio by weight to the guide. The LNPs contained Lipid A,Cholesterol, DSPC, and PEG2k-DMG in a 50:38:9:3 molar ratio,respectively, and having a N:P ratio of 6. Dosing was at 1, 0.3, 0.1, or0.03 mg/kg (n=5/group). The LNPs were prepared using the cross-flowprocedure described above and purified and concentrated using PD-10columns and Amicon centrifugal filter units, respectively. Liver editingresults were determined using primers designed to amplify the region ofinterest for NGS analysis and serum human TTR levels were measured asdescribed above. Results for liver editing are shown in FIG. 26A andserum human TTR levels in FIG. 26B-C. A dose response for both editingand serum TTR levels was evident.

In another set of experiments, humanized TTR mice were dosed with LNPformulations across a range of doses with guides G000481, G000482,G000486 and G000499. The formulations contained Cas9 mRNA (SEQ ID NO: 1)in a 1:1 ratio by weight to the guide. The LNPs contained Lipid A,Cholesterol, DSPC, and PEG2k-DMG in a 50:38:9:3 molar ratio,respectively, and had an N:P ratio of 6. Dosing was at 1, 0.3, or 0.1mg/kg (n=5/group). The LNPs were prepared using the cross-flow proceduredescribed above and purified and concentrated using PD-10 columns andAmicon centrifugal filter units, respectively. Liver editing resultswere determined using primers designed to amplify the region of interestfor NGS analysis and serum human TTR levels were measured as describedabove. Results for liver editing are shown in FIG. 27A and serum humanTTR levels in FIG. 27B-C. A dose response for both editing and serum TTRlevels was evident.

In another set of experiments, humanized TTR mice were dosed with LNPformulations across a range of doses with guides G000480, G000481,G000486, G000499 and G000502. The formulations contained Cas9 mRNA (SEQID NO: 1) in a 1:2 ratio by weight to the guide. The LNPs containedLipid A, Cholesterol, DSPC, and PEG2k-DMG in a 50:38:9:3 molar ratio,respectively, and had an N:P ratio of 6. Dosing was at 1, 0.3, or 0.1mg/kg (n=5/group). The LNPs were prepared using the cross-flow proceduredescribed above and purified and concentrated using PD-10 columns andAmicon centrifugal filter units, respectively. Liver editing resultswere determined using primers designed to amplify the region of interestfor NGS analysis and serum human TTR levels were measured as describedabove. Results for liver editing are shown in FIG. 28A and serum humanTTR levels in FIG. 28B-C. A dose response for both editing and serum TTRlevels was evident.

In separate experiments using wild type CD-1 mice, an LNP formulationcomprising guide G000502, which is cross homologous between mouse andcyno, was tested in a dose response study. The formulation containedCas9 mRNA (SEQ ID NO: 1) in a 1:1 ratio by weight to the guide. The LNPcontained Lipid A, Cholesterol, DSPC, and PEG2k-DMG in a 45:44:9:2 molarratio, respectively, and having a N:P ratio of 6. Dosing was at 1, 0.3,0.1, 0.03, or 0.01 mg/kg (n=5/group). Liver editing results weredetermined using primers designed to amplify the region of interest forNGS analysis. Results for liver editing are shown in FIG. 15A and serummouse TTR levels in FIG. 15B. A dose response for both editing and serumTTR levels was evident.

Example 9. LNP Delivery to Mice in Multiple Doses

Mice (females from Charles River Laboratory, aged approximately 6-7weeks) were dosed with an LNP formulation LNP705, prepared usingcross-flow and TFF procedures as described above containing G000282(“G282”) and Cas9 mRNA (SEQ ID NO: 2) in a 1:1 ratio by weight and atotal RNA concentration of 0.5 mg/ml. The LNP had an N:P ratio of 4.5and contained Lipid A, Cholesterol, DSPC, and PEG2k-DMG in a 45:44:9:2molar ratio, respectively. Groups were dosed either once weekly up toone, two, three, or four weeks (QWx1-4) or once monthly up to two orthree months (QMx2-3). Dosages were 0.5 mg/kg or 1 mg/kg (total RNAcontent). Control groups received a single dose on day 1 of 0.5, 1, or 2mg/kg. Each group contained 5 mice. Serum TTR was analyzed by ELISA andat necropsy the liver, spleen and muscle were each collected for NGSediting analysis. Groups are shown in Table 27. X=sacrifice andnecropsy. MPK=mg/kg.

TABLE 27 Study Groups Total Duration/ Dose Dose Dose Dose Dose NX DoseNX Dose Dose (MPK) Day Day Day Day Day Day Day Group Regimen (MPK) Given1 8 15 22 28 43 49  1 4 Week 0 (TSS 0 X X X X X Multi control) Dose/ QW× 4  2 2 Month 1 3 X X X X  3 Multi 0.5 1.5 X X X X Dose/ QM × 3  4 1Month 1 2 X X X  5 Multi 0.5 1 X X X Dose/ QM × 2  6 4 Week 1 4 X X X XX  7 Multi 0.5 2 X X X X X Dose/ QW × 4  8 3 Week 1 3 X X X X  9 Multi0.5 1.5 X X X X Dose/ QW × 3 10 2 Week 1 2 X X X 11 Multi 0.5 1 X X XDose/ QW × 2 12 Single 1 1 X X 13 Dose/ 0.5 0.5 X X 14 QW × 1 2 2 DayDay 26 32

Table 28 and FIGS. 10A-11B show serum TTR level results (% KD=%knockdown). Table 29 and FIGS. 12A-C show liver editing results.

TABLE 28 Serum TTR Results. Serum TTR Serum TTR Time Regimen Dose(μg/mL) (% KD) QWx4 TSS 1190.7 — QMx3 0.5 245.01 79.42 QMx2 0.5 776.7334.77 QWx4 0.5 347.43 70.82 QWx3 0.5 405.70 65.93 QWx2 0.5 432.25 63.70QWx1 0.5 804.06 32.47 QMx3 1 91.95 92.28 QMx2 1 176.81 85.15 QWx4 1119.52 89.96 QWx3 1 167.15 85.96 QWx2 1 130.98 89.00 QWx1 1 573.02 51.88QWx1 2 219.07 81.60

TABLE 29 Liver Editing Results. Time Regimen Dose Liver Editing (%) QWx4TSS 0.38 QMx3 0.5 48.18 QMx2 0.5 36.66 QWx4 0.5 56.03 QWx3 0.5 51.35QWx2 0.5 34.77 QWx1 0.5 24.16 QMx3 1 63.40 QMx2 1 57.37 QWx4 1 62.89QWx3 1 59.22 QWx2 1 60.12 QWx1 1 35.16 QWx1 2 60.57

The results show that it is possible to build up a cumulative dose andeffect with multiple administrations over time, including at weekly ormonthly intervals, to achieve increasing editing levels and % KD of TTR.

Example 10. RNA Cargo: Varying mRNA and gRNA Ratios

This study evaluated in vivo efficacy in mice of different ratios ofgRNA to mRNA. CleanCap™ capped Cas9 mRNAs with the ORF of SEQ ID NO: 4,HSD 5′ UTR, human albumin 3′ UTR, a Kozak sequence, and a poly-A tailwere made by IVT synthesis as indicated in Example 1 withN1-methylpseudouridine triphosphate in place of uridine triphosphate.

LNP formulations prepared from the mRNA described and G282 (SEQ ID NO:124) as described in Example 1 with Lipid A, cholesterol, DSPC, andPEG2k-DMG in a 50:38:9:3 molar ratio and with an N:P ratio of 6. ThegRNA:Cas9 mRNA weight ratios of the formulations were as shown in FIGS.19A and 19B.

For in vivo characterization, the LNPs were administered to mice at 0.1mg total RNA (mg guide RNA+mg mRNA) per kg (n=5 per group). At 7-9 dayspost-dose, animals were sacrificed, blood and the liver were collected,and serum TTR and liver editing were measured as described in Example 1.Serum TTR and liver editing results are shown in FIGS. 19A and 19B.Negative control mice were dosed with TSS vehicle.

In addition, the above LNPs were administered to mice at a constant mRNAdose of 0.05 mg mRNA per kg (n=5 per group), while varying the gRNA dosefrom 0.06 mg per kg to 0.4 mg per kg. At 7-9 days post-dose, animalswere sacrificed, blood and the liver were collected, and serum TTR andliver editing were measured. Serum TTR and liver editing results areshown in FIG. 19C and FIG. 19D. Negative control mice were dosed withTSS vehicle.

Example 11. Off-Target Analysis of TTR sgRNAs in Primary HumanHepatocyes

Off-target analysis of sgRNAs targeting TTR was performed in primaryhuman hepatocytes (PHH) as described in Example 5, with the followingmodifications. PHH were plated at a density of 33,000 cells per well oncollagen-coated 96-well plates as described in Example 1. Twenty-fourhours post plating, cells were washed with media and transfected usingLipofectamine RNAiMAX (ThermoFisher, Cat. 13778150) as described inExample 1. Cells were transfected with a lipoplex containing 100 ng Cas9mRNA, immediately followed by the addition of another lipoplexcontaining 25 nM of the sgRNA and 12.5 nM of the donor oligo (0.3μL/well). Cells were lysed 48 hours post-transfection and gDNA wasextracted and analyzed as further described in Example 5. The data isgraphically represented in FIG. 20.

Table 30 shows the number of off-target integration sites detected inPHH, and compares to the number of sites that were detected in theHekCas9 cells used in Example 5. Fewer sites were detected in PHH forevery guide tested as compared to the HekCas9 cell line, with no uniquesites detected in PHH alone.

TABLE 30 Number of off-target integration sites detected for TTR sgRNAsin PHH via an oligo insertion based assay # Sites in # HekCas9 cellsGUIDE ID Sites in PHH (Example 5) G000480 2 11 G000481 0 3 G000482 2 13G000483 0 5 G000484 0 7 G000485 3 22 G000486 0 12 G000487 0 14 G000488 00 G000489 2 19 G000490 0 12 G000491 7 28 G000492 5 97 G000493 1 7G000494 0 4 G000495 1 13 G000496 0 1 G000497 3 26 G000498 19 82 G0004991 4 G000500 12 46 G000501 0 4 G000567 0 9 G000568 11 936 G000570 1 19G000571 1 16 G000572 2 15

Following the identification of potential off-target sites in PHH viathe oligo insertion assay, certain potential sites were furtherevaluated by targeted amplicon sequencing, e.g., as described in Example6. In addition to the potential off-target sites identified by the oligoinsertion strategy, additional potential off-target sites identified byin silico prediction were included in the analysis.

To this end, PHH were treated with LNPs comprising 100 ng of Cas9 mRNA(SEQ ID NO:1) and the gRNA of interest at 14.68 nM (in a 1:1 ratio byweight), as described in Example 4. The LNPs were prepared using thecross-flow procedure described above and purified and concentrated usingPD-10 columns and Amicon centrifugal filter units, respectively. TheLNPs were formulated with an N:P ratio of 6.0 and contained Lipid A,Cholesterol, DSPC, and PEG2k-DMG in a 50:38:9:2 molar ratio,respectively. Following LNP treatment, isolated genomic DNA was analyzedby NGS (e.g., as described in Examples 1 and 6) to determine whetherindels could be detected at the potential off-target site, which wouldbe indicative of a Cas9-mediated cleavage event. Tables 31 and 32 showthe potential off-target sites that were evaluated for the gRNAs G000480and G000486, respectively.

As shown in FIGS. 21A-B and 22A-B and Table 33 below, indels weredetected at low levels for only two of the potential off-target sitesidentified by the oligo insertion assay for G000480, and only one forG000486. No indels were detected at any of the in silico predicted sitesfor either guide. Further, indels were only detected at these sitesusing a near-saturating dose of LNP, as the indel rates observed at theon-target sites for G000480 and G000486 were ˜97% and ˜91%, respectively(See Table 33). The genomic coordinates of these sites are also reportedin Tables 31 and 32, and each correspond to sequences that do not codefor any protein.

A dose response assay was then performed in order to determine thehighest dose of LNP in which no off-targets were detected. PHH weretreated with LNPs comprising either G000480 or G000486 as described inExample 4. The doses ranged across 11 points with respect to gRNAconcentration (0.001 nM, 0.002 nM, 0.007 nM, 0.02 nM, 0.06 nM, 0.19 nM,0.57 nM, 1.72 nM, 5.17 nM, 15.51 nM, and 46.55 nM). As represented bythe dashed vertical line in FIGS. 21A-B and 22A-B, the highestconcentrations (with respect to the concentration of gRNA) at which thepotential off-target sites were no longer detected for G000480 andG000486 were 0.57 nM and 15.51 nM, respectively, which resulted inon-target indel rates of 84.60% and 89.50%, respectively.

TABLE 31 Identified potential off target sites via insertion detectionand in silico prediction for G000480 evaluated via targeted ampliconsequencing Off-target Chromosomal Coordinates GUIDE ID (OT) Site IDAssay Used (hg38) Strand G000480 INS-OT.1 Insertion Detection chr7:94767406-94767426 + G000480 INS-OT.2 Insertion Detection chr2:192658562-192658582 + G000480 INS-OT.3 Insertion Detection chr7:4834390-4834410 + G000480 INS-OT.4 Insertion Detection chr20:9216118-9216138 − G000480 INS-OT.5 Insertion Detection chr10:12547071-12547091 + G000480 INS-OT.6 Insertion Detection chr6:168377978-168377998 − G000480 INS-OT.7 Insertion Detection chr12:114144669-114144689 − G000480 INS-OT.8 Insertion Detection chr10:7376755-7376775 + G000480 INS-OT.9 Insertion Detection chr2:52950299-52950319 + G000480 INS-OT.10 Insertion Detection chr8:56579165-56579185 − G000480 INS-OT.11 Insertion Detection chr1:189992255-189992275 + G000480 PRED-OT.1 in silico predictionchr10:12547071-12547091 + G000480 PRE-DOT.2 in silico prediction chrX:119702782-119702802 + G000480 PRED-OT.3 in silico prediction chr1:116544586-116544606 + G000480 PRED-OT.4 in silico prediction chr6:88282884-88282904 + G000480 PRED-OT.6 in silico prediction chr5:121891868-121891888 + G000480 PRED-OT.7 in silico prediction chr3:52544945-52544965 + G000480 PRED-OT.8 in silico prediction chr15:36949639-36949659 + G000480 PRED-OT.9 in silico prediction chr5:33866486-33866506 + G000480 PRED-OT.10 in silico prediction chr5:159755754-159755774 + G000480 PRED-OT.11 in silico prediction chr5:31349859-31349879 + G000480 PRED-OT.12 in silico prediction chr11:79485652-79485672 + G000480 PRED-OT.13 in silico prediction chr15:29448864-29448884 + G000480 PRED-OT.14 in silico prediction chr5:171153565-171153585 + G000480 PRED-OT.15 in silico prediction chr9:84855273-84855293 + G000480 PRED-OT.16 in silico prediction chr6:159953060-159953080 + G000480 PRED-OT.17 in silico prediction chr16:51849024-51849044 + G000480 PRED-OT.18 in silico prediction chr3:24108809-24108829 + G000480 PRED-OT.19 in silico prediction chr18:41118310-41118330 + G000480 PRED-OT.20 in silico prediction chr10:108975241-108975261 + G000480 PREDO-T.21 in silico prediction chr1:44683633-44683653 + G000480 PRED-OT.22 in silico prediction chr2:196214849-196214869 + G000480 PRED-OT.23 in silico prediction chr9:117353544-117353564 + G000480 PRED-OT.24 in silico prediction chr1:55583322-55583342 + G000480 PRED-OT.25 in silico prediction chr12:28246827-28246847 + G000480 PRED-OT.26 in silico prediction chr4:54545361-54545381 + G000480 PRED-OT.27 in silico prediction chr13:22364836-22364856 + G000480 PRED-OT.28 in silico prediction chr13:80816049-80816069 + G000480 PRED-OT.29 in silico prediction chr7:39078622-39078642 + G000480 PRED-OT.30 in silico prediction chr2:59944386-59944406 + “INS-OT.N” refers to an off-target site ID detectedby oligo insertion, where N is an integer specified above; “PRED-OT.N”refers to an off-target site ID predicted via in silico methods, where Nis an integer specified above.

TABLE 32 Identified potential off target sites via insertion detectionand in silico prediction for G000486 evaluated via targeted ampliconsequencing Off-target Chromosomal Coordinates GUIDE ID (OT) Site IDAssay Used (hg38) Strand G000486 INS-OT.1 Insertion Detection chr14:77332157-77332177 + G000486 INS-OT.2 Insertion Detection chr14:54672059-54672079 − G000486 INS-OT.3 Insertion Detection chr4:108513169-108513189 − G000486 INS-OT.4 Insertion Detection chr5:91397023-91397043 − G000486 INS-OT.5 Insertion Detectionchr9:116626135-116626155 − G000486 INS-OT.6 Insertion Detection chr6:73201226-73201246 + G000486 INS-OT.7 Insertion Detection chr16:89368352-89368372 − G000486 INS-OT.8 Insertion Detection chr7:56308371-56308391 − G000486 INS-OT.9 Insertion Detectionchr21:43605667-43605687 + G000486 INS-OT.10 Insertion Detection chr5:26758030-26758050 + G000486 INS-OT.11 Insertion Detection chr17:30656428-30656448 + G000486 INS-OT.12 Insertion Detection chr8:130486452-130486472 + G000486 PRED-OT.1 in silico prediction chr11:44707064-44707084 + G000486 PRED-OT.2 in silico prediction chr5:50775396-50775416 + G000486 PRED-OT.3 in silico prediction chr4:141623949-141623969 + G000486 PRED-OT.4 in silico prediction chr1:223481186-223481206 + G000486 PRED-OT.5 in silico prediction chr6:39951487-39951507 + G000486 PRED-OT.6 in silico prediction chrY:5456047-5456067 + G000486 PRED-OT.8 in silico prediction chr6:129868719-129868739 + G000486 PRED-OT.9 in silico prediction chrX:80450312-80450332 + G000486 PRED-OT.10 in silico prediction chr7:27256771-27256791 + G000486 PRED-OT.11 in silico prediction chr3:181416528-181416548 + G000486 PRED-OT12 in silico prediction chr7:146425020-146425040 + G000486 PRED-OT.13 in silico prediction chr3:16980977-16980997 + G000486 PRED-OT.14 in silico prediction chr7:118161002-118161022 + G000486 PRED-OT.15 in silico prediction chr6:102220539-102220559 + G000486 PRED-OT.16 in silico prediction chr12:127278991-127279011 + G000486 PRED-OT.17 in silico prediction chr2:67686631-67686651 + G000486 PRED-OT.18 in silico prediction chr1:114467665-114467685 + G000486 PRED-OT.19 in silico prediction chr3:194514436-194514456 + G000486 PRED-OT.20 in silico prediction chr14:31767581-31767601 + G000486 PRED-OT.21 in silico prediction chr16:28706209-28706229 + G000486 PRED-OT.22 in silico prediction chr8:110526279-110526299 + G000486 PRED-OT.23 in silico prediction chr19:2899814-2899834 + G000486 PRED-OT.25 in silico prediction chr3:130760261-130760281 + G000486 PRED-OT.26 in silico prediction chr11:2506046-2506066 + G000486 PRED-OT.27 in silico prediction chr2:153918318-153918338 + G000486 PRED-OT.28 in silico prediction chr14:40590226-40590246 + G000486 PRED-OT.29 in silico prediction chr18:806650-806670 + G000486 PRED-OT.30 in silico prediction chr2:117707480-117707500 + “INS-OT.N” refers to an off-target site IDdetected by oligo insertion, where N is an integer specified above;“PRED-OT.N” refers to an off-target site ID predicted via in silicomethods, where N is an integer specified.

TABLE 33 Detected Off Target sites in PHH treated with LNP containing100 ng mRNA and 31.03 nM gRNA Indel Frequency (using LNP with 100 ngCas9 Indel Off-target mRNA and 14.68 Frequency GUIDE ID (OT) Site IDSite Type nM gRNA) std. dev. G000480 n/a On-Target 97.33% 1.10% G000480INS-OT.2 Off-Target 1.43% 0.40% G000480 INS-OT.4 Off-Target 0.97% 0.25%G000486 n/a On-Target 91.33% 1.97% G000486 INS-OT.4 Off-Target 0.47%0.06%

Example 12. LNP Delivery to Humanized Mouse Model of ATTR

A well-established humanized transgenic mouse model of hereditary ATTRamyloidosis that expresses the V30M pathogenic mutant form of human TTRprotein was used in this Example. This mouse model recapitulates the TTRdeposition phenotype in tissues observed in ATTR patients, includingwithin the peripheral nervous system and gastrointestinal (GI) tract(See Santos et al., Neurobiol Aging. 2010 February; 31(2):280-9).

Mice (aged approximately 4-5 months) were dosed with LNP formulationsprepared using the cross-flow and TFF procedures as described inExample 1. The LNPs were formulated with an N:P ratio of 6.0 andcontained Lipid A, Cholesterol, DSPC, and PEG2k-DMG in a 50:38:9:2 molarratio, respectively. The LNPs contained Cas9 mRNA (SEQ ID NO: 1) andeither G000481 (“G481”) or a non-targeting control guide G000395(“G395”; SEQ ID NO: 273), in a 1:1 ratio of gRNA:mRNA by weight.

Mice were injected via the lateral tail vein as described in Example 1with a single 1 mg/kg (of total RNA content) dose of LNP with ann=10/group. At 8 weeks post treatment, the mice were euthanized forsample collection. Human TTR protein levels were measured in serum andcerebrospinal fluid (CSF) by ELISA as previously described by Butler etal., Amyloid. 2016 June; 23(2):109-18. Liver tissue was assayed forediting levels as described in Example 1. Other tissues (stomach, colon,sciatic nerve, dorsal root ganglion (DRG)) were collected and processedfor semi-quantitative immunohistochemistry as previously described byGonçalves et al., Amyloid. 2014 September; 21(3): 175-184. Statisticalanalysis for the immunohistochemistry data was performed using MannWhitney test with a p-value<0.0001.

As shown in FIG. 23A-B, robust editing (49.4%) of TTR was observed inlivers of the humanized mice following the single dose of LNP comprisingG481, with no editing detected in the control group. Analysis of theediting events demonstrated that 96.8% of the events were insertions,with the remainder deletions.

As shown in FIG. 24A-B, TTR protein levels were decreased in plasma butnot in CSF from the treated mice, with greater than 99% knockdown of TTRplasma levels observed (p<0.001).

The near complete knockdown of TTR observed in the plasma of treatedanimals correlated with the clearance of TTR protein amyloid depositionin the assayed tissues. As shown in FIG. 25, control mice exhibitedamyloid staining in tissues which resembles the pathophysiology observedin human subjects with ATTR. Decreasing circulating TTR by editing theHuTTR V30M locus resulted in a dramatic decrease of amyloid depositionin tissues. Approximately 85% or better reduction in TTR staining wasobserved across the treated tissues 8 weeks post-treatment (FIG. 25).

Example 13. TTR mRNA Knockdown in Primary Human Hepatocytes (PHH)

In one experiment, PHH were cultured and treated with LNPs comprisingCas9 mRNA (SEQ ID NO:1) and a gRNA of interest (See FIG. 29, Table 34),as described in Example 4. The LNPs were prepared using the cross-flowprocedure described above and purified and concentrated using PD-10columns and Amicon centrifugal filter units, respectively. The LNPs wereformulated with an N:P ratio of 6.0 and contained Lipid A, Cholesterol,DSPC, and PEG2k-DMG in a 50:38:9:2 molar ratio, respectively. The LNPscomprised a gRNA:mRNA ratio of 1:2, and the cells were treated at a doseof 300 ng (with respect to the amount of mRNA cargo delivered).

Ninety-six (96) hours following LNP treatment (with biologicaltriplicates for each condition), mRNA was purified from PHH cells usingthe Dynabeads mRNA DIRECT Kit (ThermoFisher Scientific) according to themanufacturer's protocol. Reverse Transcription (RT) was performed withMaxima reverse transcriptase (ThermoFisher Scientific) and a poly-dTprimer. The resulting cDNA was purified with Ampure XP Beads(Agencourt). For Quantitative PCR, 2% of the purified cDNA was amplifiedwith Taqman Fast Advanced Mastermix and 3 Taqman probe sets, TTR (AssayID: Hs00174914_m1), GAPDH (Assay ID: Hs02786624_g1), and PPIB (Assay ID:Hs00168719_m1). The assays were run on the QuantStudio 7 Flex Real TimePCR System according to the manufacturer's instructions (LifeTechnologies). Relative expression of TTR mRNA was calculated bynormalizing to the endogenous controls (GAPDH and PPIB) individually,and then averaged.

As shown in FIG. 29 and reproduced numerically in Table 34 below, eachof the LNP formulations tested resulted in knockdown of TTR mRNA, ascompared to the negative (untreated) control. The groups in FIG. 29 andTable 34 are identified by the gRNA ID used in each LNP preparation.Relative expression of TTR mRNA is plotted in FIG. 29, whereas thepercent knockdown of TTR mRNA is provided in Table 34.

TABLE 34 GUIDE ID Avg % Knockdown Std Dev G000480 95.19 1.68 G00048191.39 2.39 G000482 82.31 4.51 G000483 68.78 13.45 G000484 75.22 9.05G000488 92.77 3.76 G000489 91.85 2.77 G000490 78.34 5.76 G000493 87.534.54 G000494 91.15 3.63 G000499 91.38 1.71 G000500 92.90 3.15 G00056790.89 5.39 G000568 53.44 20.20 G000570 93.38 2.66 G000571 96.17 2.07G000572 55.92 24.53

In a separate experiment, TTR mRNA knockdown was evaluated followingtreatment with LNPs comprising G000480, G000486, and G000502. The LNPswere formulated and PHH were cultured and treated with the LNPs, each asdescribed in the experiment above in this Example with the exceptionthat the cells were treated at a dose of 100 ng (with respect to theamount of mRNA cargo delivered).

Ninety-six (96) hours following LNP treatment (single treatment for eachcondition), mRNA was purified from PHH cells using the Dynabeads mRNADIRECT Kit (ThermoFisher Scientific) according to the manufacturer'sprotocol. Reverse Transcription (RT) was performed with the HighCapacity cDNA Reverse Transcription Kit (ThermoFisher Scientific)according to the manufacturer's instructions. For Quantitative PCR, 2%of the cDNA was amplified with Taqman Fast Advanced Mastermix and 3Taqman probe sets, TTR (Assay ID: Hs00174914_m1), GAPDH (Assay ID:Hs02786624_g1), and PPIB (Assay ID: Hs00168719_m1). The assays were runon the QuantStudio 7 Flex Real Time PCR System according to themanufacturer's instructions (Life Technologies). Relative expression ofTTR mRNA was calculated by normalizing to the endogenous controls (GAPDHand PPIB) individually, and then averaged.

As shown in FIG. 30 and reproduced numerically in Table 35 below, eachof the LNP formulations tested resulted in knockdown of TTR mRNA, ascompared to the negative (untreated) control. The groups in FIG. 30 andTable 35 are identified by the gRNA ID used in each LNP preparation.Relative expression of TTR mRNA is plotted in FIG. 30, whereas thepercent knockdown of TTR mRNA is provided in Table 35.

TABLE 35 GUIDE ID Avg % Knockdown Std Dev G000480 95.61 0.92 G00048697.36 0.63 G000502 90.94 2.63

Sequence Table SEQ Description Sequence ID No. Cas9GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGTGTGTCGTT 1 transcriptGCAGGCCTTATTCGGATCCGCCACCATGGACAAGAAGTACAGCATCGGACT with 5′ UTRGGACATCGGAACAAACAGCGTCGGATGGGCAGTCATCACAGACGAATACAA of HSD, ORFGGTCCCGAGCAAGAAGTTCAAGGTCCTGGGAAACACAGACAGACACAGCAT correspondingCAAGAAGAACCTGATCGGAGCACTGCTGTTCGACAGCGGAGAAACAGCAGA to SEQ IDAGCAACAAGACTGAAGAGAACAGCAAGAAGAAGATACACAAGAAGAAAGAA NO: 204,CAGAATCTGCTACCTGCAGGAAATCTTCAGCAACGAAATGGCAAAGGTCGA KozakCGACAGCTTCTTCCACAGACTGGAAGAAAGCTTCCTGGTCGAAGAAGACAA sequence,GAAGCACGAAAGACACCCGATCTTCGGAAACATCGTCGACGAAGTCGCATA and 3′ UTRCCACGAAAAGTACCCGACAATCTACCACCTGAGAAAGAAGCTGGTCGACAG of ALBCACAGACAAGGCAGACCTGAGACTGATCTACCTGGCACTGGCACACATGATCAAGTTCAGAGGACACTTCCTGATCGAAGGAGACCTGAACCCGGACAACAGCGACGTCGACAAGCTGTTCATCCAGCTGGTCCAGACATACAACCAGCTGTTCGAAGAAAACCCGATCAACGCAAGCGGAGTCGACGCAAAGGCAATCCTGAGCGCAAGACTGAGCAAGAGCAGAAGACTGGAAAACCTGATCGCACAGCTGCCGGGAGAAAAGAAGAACGGACTGTTCGGAAACCTGATCGCACTGAGCCTGGGACTGACACCGAACTTCAAGAGCAACTTCGACCTGGCAGAAGACGCAAAGCTGCAGCTGAGCAAGGACACATACGACGACGACCTGGACAACCTGCTGGCACAGATCGGAGACCAGTACGCAGACCTGTTCCTGGCAGCAAAGAACCTGAGCGACGCAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAAATCACAAAGGCACCGCTGAGCGCAAGCATGATCAAGAGATACGACGAACACCACCAGGACCTGACACTGCTGAAGGCACTGGTCAGACAGCAGCTGCCGGAAAAGTACAAGGAAATCTTCTTCGACCAGAGCAAGAACGGATACGCAGGATACATCGACGGAGGAGCAAGCCAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAAGATGGACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAAGACCTGCTGAGAAAGCAGAGAACATTCGACAACGGAAGCATCCCGCACCAGATCCACCTGGGAGAACTGCACGCAATCCTGAGAAGACAGGAAGACTTCTACCCGTTCCTGAAGGACAACAGAGAAAAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTACGTCGGACCGCTGGCAAGAGGAAACAGCAGATTCGCATGGATGACAAGAAAGAGCGAAGAAACAATCACACCGTGGAACTTCGAAGAAGTCGTCGACAAGGGAGCAAGCGCACAGAGCTTCATCGAAAGAATGACAAACTTCGACAAGAACCTGCCGAACGAAAAGGTCCTGCCGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTACAACGAACTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCCGGCATTCCTGAGCGGAGAACAGAAGAAGGCAATCGTCGACCTGCTGTTCAAGACAAACAGAAAGGTCACAGTCAAGCAGCTGAAGGAAGACTACTTCAAGAAGATCGAATGCTTCGACAGCGTCGAAATCAGCGGAGTCGAAGACAGATTCAACGCAAGCCTGGGAACATACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGACAACGAAGAAAACGAAGACATCCTGGAAGACATCGTCCTGACACTGACACTGTTCGAAGACAGAGAAATGATCGAAGAAAGACTGAAGACATACGCACACCTGTTCGACGACAAGGTCATGAAGCAGCTGAAGAGAAGAAGATACACAGGATGGGGAAGACTGAGCAGAAAGCTGATCAACGGAATCAGAGACAAGCAGAGCGGAAAGACAATCCTGGACTTCCTGAAGAGCGACGGATTCGCAAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACATTCAAGGAAGACATCCAGAAGGCACAGGTCAGCGGACAGGGAGACAGCCTGCACGAACACATCGCAAACCTGGCAGGAAGCCCGGCAATCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAACTGGTCAAGGTCATGGGAAGACACAAGCCGGAAAACATCGTCATCGAAATGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAATGAAGAGAATCGAAGAAGGAATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACACCCGGTCGAAAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCTGCAGAACGGAAGAGACATGTACGTCGACCAGGAACTGGACATCAACAGACTGAGCGACTACGACGTCGACCACATCGTCCCGCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTCCTGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGTCCCGAGCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGACAGCTGCTGAACGCAAAGCTGATCACACAGAGAAAGTTCGACAACCTGACAAAGGCAGAGAGAGGAGGACTGAGCGAACTGGACAAGGCAGGATTCATCAAGAGACAGCTGGTCGAAACAAGACAGATCACAAAGCACGTCGCACAGATCCTGGACAGCAGAATGAACACAAAGTACGACGAAAACGACAAGCTGATCAGAGAAGTCAAGGTCATCACACTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGACTTCCAGTTCTACAAGGTCAGAGAAATCAACAACTACCACCACGCACACGACGCATACCTGAACGCAGTCGTCGGAACAGCACTGATCAAGAAGTACCCGAAGCTGGAAAGCGAATTCGTCTACGGAGACTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGAGCGAACAGGAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAAGACAGAAATCACACTGGCAAACGGAGAAATCAGAAAGAGACCGCTGATCGAAACAAACGGAGAAACAGGAGAAATCGTCTGGGACAAGGGAAGAGACTTCGCAACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCGTCAAGAAGACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAGCATCCTGCCGAAGAGAAACAGCGACAAGCTGATCGCAAGAAAGAAGGACTGGGACCCGAAGAAGTACGGAGGATTCGACAGCCCGACAGTCGCATACAGCGTCCTGGTCGTCGCAAAGGTCGAAAAGGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGCTGGGAATCACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGATCGACTTCCTGGAAGCAAAGGGATACAAGGAAGTCAAGAAGGACCTGATCATCAAGCTGCCGAAGTACAGCCTGTTCGAACTGGAAAACGGAAGAAAGAGAATGCTGGCAAGCGCAGGAGAACTGCAGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGTCAACTTCCTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCTGTTCGTCGAACAGCACAAGCACTACCTGGACGAAATCATCGAACAGATCAGCGAATTCAGCAAGAGAGTCATCCTGGCAGACGCAAACCTGGACAAGGTCCTGAGCGCATACAACAAGCACAGAGACAAGCCGATCAGAGAACAGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTGGGAGCACCGGCAGCATTCAAGTACTTCGACACAACAATCGACAGAAAGAGATACACAAGCACAAAGGAAGTCCTGGACGCAACACTGATCCACCAGAGCATCACAGGACTGTACGAAACAAGAATCGACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAAGCCCGAAGAAGAAGAGAAAGGTCTAGCTAGCCATCACATTTAAAAGCATCTCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAATAGCTTATTCATCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAACATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAATAAAAAATGGAAAGAACCTCGAG Cas9GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGTGTGTCGTT 2 transcriptGCAGGCCTTATTCGGATCCATGCCTAAGAAAAAGCGGAAGGTCGACGGGGA comprisingTAAGAAGTACTCAATCGGGCTGGATATCGGAACTAATTCCGTGGGTTGGGC Cas9 ORFAGTGATCACGGATGAATACAAAGTGCCGTCCAAGAAGTTCAAGGTCCTGGG correspondingGAACACCGATAGACACAGCATCAAGAAAAATCTCATCGGAGCCCTGCTGTT to SEQ IDTGACTCCGGCGAAACCGCAGAAGCGACCCGGCTCAAACGTACCGCGAGGCG NO: 205ACGCTACACCCGGCGGAAGAATCGCATCTGCTATCTGCAAGAGATCTTTTC using codonsGAACGAAATGGCAAAGGTCGACGACAGCTTCTTCCACCGCCTGGAAGAATC withTTTCCTGGTGGAGGAGGACAAGAAGCATGAACGGCATCCTATCTTTGGAAA generallyCATCGTCGACGAAGTGGCGTACCACGAAAAGTACCCGACCATCTACCATCT highGCGGAAGAAGTTGGTTGACTCAACTGACAAGGCCGACCTCAGATTGATCTA expressionCTTGGCCCTCGCCCATATGATCAAATTCCGCGGACACTTCCTGATCGAAGG in humansCGATCTGAACCCTGATAACTCCGACGTGGATAAGCTTTTCATTCAACTGGTGCAGACCTACAACCAACTGTTCGAAGAAAACCCAATCAATGCTAGCGGCGTCGATGCCAAGGCCATCCTGTCCGCCCGGCTGTCGAAGTCGCGGCGCCTCGAAAACCTGATCGCACAGCTGCCGGGAGAGAAAAAGAACGGACTTTTCGGCAACTTGATCGCTCTCTCACTGGGACTCACTCCCAATTTCAAGTCCAATTTTGACCTGGCCGAGGACGCGAAGCTGCAACTCTCAAAGGACACCTACGACGACGACTTGGACAATTTGCTGGCACAAATTGGCGATCAGTACGCGGATCTGTTCCTTGCCGCTAAGAACCTTTCGGACGCAATCTTGCTGTCCGATATCCTGCGCGTGAACACCGAAATAACCAAAGCGCCGCTTAGCGCCTCGATGATTAAGCGGTACGACGAGCATCACCAGGATCTCACGCTGCTCAAAGCGCTCGTGAGACAGCAACTGCCTGAAAAGTACAAGGAGATCTTCTTCGACCAGTCCAAGAATGGGTACGCAGGGTACATCGATGGAGGCGCTAGCCAGGAAGAGTTCTATAAGTTCATCAAGCCAATCCTGGAAAAGATGGACGGAACCGAAGAACTGCTGGTCAAGCTGAACAGGGAGGATCTGCTCCGGAAACAGAGAACCTTTGACAACGGATCCATTCCCCACCAGATCCATCTGGGTGAGCTGCACGCCATCTTGCGGCGCCAGGAGGACTTTTACCCATTCCTCAAGGACAACCGGGAAAAGATCGAGAAAATTCTGACGTTCCGCATCCCGTATTACGTGGGCCCACTGGCGCGCGGCAATTCGCGCTTCGCGTGGATGACTAGAAAATCAGAGGAAACCATCACTCCTTGGAATTTCGAGGAAGTTGTGGATAAGGGAGCTTCGGCACAAAGCTTCATCGAACGAATGACCAACTTCGACAAGAATCTCCCAAACGAGAAGGTGCTTCCTAAGCACAGCCTCCTTTACGAATACTTCACTGTCTACAACGAACTGACTAAAGTGAAATACGTTACTGAAGGAATGAGGAAGCCGGCCTTTCTGTCCGGAGAACAGAAGAAAGCAATTGTCGATCTGCTGTTCAAGACCAACCGCAAGGTGACCGTCAAGCAGCTTAAAGAGGACTACTTCAAGAAGATCGAGTGTTTCGACTCAGTGGAAATCAGCGGGGTGGAGGACAGATTCAACGCTTCGCTGGGAACCTATCATGATCTCCTGAAGATCATCAAGGACAAGGACTTCCTTGACAACGAGGAGAACGAGGACATCCTGGAAGATATCGTCCTGACCTTGACCCTTTTCGAGGATCGCGAGATGATCGAGGAGAGGCTTAAGACCTACGCTCATCTCTTCGACGATAAGGTCATGAAACAACTCAAGCGCCGCCGGTACACTGGTTGGGGCCGCCTCTCCCGCAAGCTGATCAACGGTATTCGCGATAAACAGAGCGGTAAAACTATCCTGGATTTCCTCAAATCGGATGGCTTCGCTAATCGTAACTTCATGCAATTGATCCACGACGACAGCCTGACCTTTAAGGAGGACATCCAAAAAGCACAAGTGTCCGGACAGGGAGACTCACTCCATGAACACATCGCGAATCTGGCCGGTTCGCCGGCGATTAAGAAGGGAATTCTGCAAACTGTGAAGGTGGTCGACGAGCTGGTGAAGGTCATGGGACGGCACAAACCGGAGAATATCGTGATTGAAATGGCCCGAGAAAACCAGACTACCCAGAAGGGCCAGAAAAACTCCCGCGAAAGGATGAAGCGGATCGAAGAAGGAATCAAGGAGCTGGGCAGCCAGATCCTGAAAGAGCACCCGGTGGAAAACACGCAGCTGCAGAACGAGAAGCTCTACCTGTACTATTTGCAAAATGGACGGGACATGTACGTGGACCAAGAGCTGGACATCAATCGGTTGTCTGATTACGACGTGGACCACATCGTTCCACAGTCCTTTCTGAAGGATGACTCGATCGATAACAAGGTGTTGACTCGCAGCGACAAGAACAGAGGGAAGTCAGATAATGTGCCATCGGAGGAGGTCGTGAAGAAGATGAAGAATTACTGGCGGCAGCTCCTGAATGCGAAGCTGATTACCCAGAGAAAGTTTGACAATCTCACTAAAGCCGAGCGCGGCGGACTCTCAGAGCTGGATAAGGCTGGATTCATCAAACGGCAGCTGGTCGAGACTCGGCAGATTACCAAGCACGTGGCGCAGATCTTGGACTCCCGCATGAACACTAAATACGACGAGAACGATAAGCTCATCCGGGAAGTGAAGGTGATTACCCTGAAAAGCAAACTTGTGTCGGACTTTCGGAAGGACTTTCAGTTTTACAAAGTGAGAGAAATCAACAACTACCATCACGCGCATGACGCATACCTCAACGCTGTGGTCGGTACCGCCCTGATCAAAAAGTACCCTAAACTTGAATCGGAGTTTGTGTACGGAGACTACAAGGTCTACGACGTGAGGAAGATGATAGCCAAGTCCGAACAGGAAATCGGGAAAGCAACTGCGAAATACTTCTTTTACTCAAACATCATGAACTTTTTCAAGACTGAAATTACGCTGGCCAATGGAGAAATCAGGAAGAGGCCACTGATCGAAACTAACGGAGAAACGGGCGAAATCGTGTGGGACAAGGGCAGGGACTTCGCAACTGTTCGCAAAGTGCTCTCTATGCCGCAAGTCAATATTGTGAAGAAAACCGAAGTGCAAACCGGCGGATTTTCAAAGGAATCGATCCTCCCAAAGAGAAATAGCGACAAGCTCATTGCACGCAAGAAAGACTGGGACCCGAAGAAGTACGGAGGATTCGATTCGCCGACTGTCGCATACTCCGTCCTCGTGGTGGCCAAGGTGGAGAAGGGAAAGAGCAAAAAGCTCAAATCCGTCAAAGAGCTGCTGGGGATTACCATCATGGAACGATCCTCGTTCGAGAAGAACCCGATTGATTTCCTCGAGGCGAAGGGTTACAAGGAGGTGAAGAAGGATCTGATCATCAAACTCCCCAAGTACTCACTGTTCGAACTGGAAAATGGTCGGAAGCGCATGCTGGCTTCGGCCGGAGAACTCCAAAAAGGAAATGAGCTGGCCTTGCCTAGCAAGTACGTCAACTTCCTCTATCTTGCTTCGCACTACGAAAAACTCAAAGGGTCACCGGAAGATAACGAACAGAAGCAGCTTTTCGTGGAGCAGCACAAGCATTATCTGGATGAAATCATCGAACAAATCTCCGAGTTTTCAAAGCGCGTGATCCTCGCCGACGCCAACCTCGACAAAGTCCTGTCGGCCTACAATAAGCATAGAGATAAGCCGATCAGAGAACAGGCCGAGAACATTATCCACTTGTTCACCCTGACTAACCTGGGAGCCCCAGCCGCCTTCAAGTACTTCGATACTACTATCGATCGCAAAAGATACACGTCCACCAAGGAAGTTCTGGACGCGACCCTGATCCACCAAAGCATCACTGGACTCTACGAAACTAGGATCGATCTGTCGCAGCTGGGTGGCGATTGATAGTCTAGCCATCACATTTAAAAGCATCTCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAATAGCTTATTCATCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAACATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAATAAAAA ATGGAAAGAACCTCGAGmodified mN*mN*mN*NNNNNNNNNNNNNNNNNGUUUUAGAmGmCmUmAmGmAmAmAm 3 sgRNAUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA sequencemAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU (″N″ may beany natural or non- natural nucleotide) 30/30/39AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCGAAAAAAAAAAAAAAAAAA 3 poly-AAAAAAAAAAAAACCGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA sequence AAACR003335 CUGCUCCUCCUCUGCCUUGC 5 gRNA targeting Human TTR (Exon 1)CR003336 CCUCCUCUGCCUUGCUGGAC 6 gRNA targeting Human TTR (Exon 1)CR003337 CCAGUCCAGCAAGGCAGAGG 7 gRNA targeting Human TTR (Exon 1)CR003338 AUACCAGUCCAGCAAGGCAG 8 gRNA targeting Human TTR (Exon 1)CR003339 ACACAAAUACCAGUCCAGCA 9 gRNA targeting Human TTR (Exon 1)CR003340 UGGACUGGUAUUUGUGUCUG 10 gRNA targeting Human TTR (Exon 1)CR003341 CUGGUAUUUGUGUCUGAGGC 11 gRNA targeting Human TTR (Exon 1)CR003342 CUUCUCUACACCCAGGGCAC 12 gRNA targeting Human TTR (Exon 2)CR003343 CAGAGGACACUUGGAUUCAC 13 gRNA targeting Human TTR (Exon 2)CR003344 UUUGACCAUCAGAGGACACU 14 gRNA targeting Human TTR (Exon 2)CR003345 UCUAGAACUUUGACCAUCAG 15 gRNA targeting Human TTR (Exon 2)CR003346 AAAGUUCUAGAUGCUGUCCG 16 gRNA targeting Human TTR (Exon 2)CR003347 CAUUGAUGGCAGGACUGCCU 17 gRNA targeting Human TTR (Exon 2)CR003348 AGGCAGUCCUGCCAUCAAUG 18 gRNA targeting Human TTR (Exon 2)CR003349 UGCACGGCCACAUUGAUGGC 19 gRNA targeting Human TTR (Exon 2)CR003350 CACAUGCACGGCCACAUUGA 20 gRNA targeting Human TTR (Exon 2)CR003351 AGCCUUUCUGAACACAUGCA 21 gRNA targeting Human TTR (Exon 2)CR003352 GAAAGGCUGCUGAUGACACC 22 gRNA targeting Human TTR (Exon 2)CR003353 AAAGGCUGCUGAUGACACCU 23 gRNA targeting Human TTR (Exon 2)CR003354 ACCUGGGAGCCAUUUGCCUC 24 gRNA targeting Human TTR (Exon 2)CR003355 CCCAGAGGCAAAUGGCUCCC 25 gRNA targeting Human TTR (Exon 2)CR003356 GCAACUUACCCAGAGGCAAA 26 gRNA targeting Human TTR (Exon 2)CR003357 UUCUUUGGCAACUUACCCAG 27 gRNA targeting Human TTR (Exon 2)CR003358 AUGCAGCUCUCCAGACUCAC 28 gRNA targeting Human TTR (Exon 3)CR003359 AGUGAGUCUGGAGAGCUGCA 29 gRNA targeting Human TTR (Exon 3)CR003360 GUGAGUCUGGAGAGCUGCAU 30 gRNA targeting Human TTR (Exon 3)CR003361 GCUGCAUGGGCUCACAACUG 31 gRNA targeting Human TTR (Exon 3)CR003362 GCAUGGGCUCACAACUGAGG 32 gRNA targeting Human TTR (Exon 3)CR003363 ACUGAGGAGGAAUUUGUAGA 33 gRNA targeting Human TTR (Exon 3)CR003364 CUGAGGAGGAAUUUGUAGAA 34 gRNA targeting Human TTR (Exon 3)CR003365 UGUAGAAGGGAUAUACAAAG 35 gRNA targeting Human TTR (Exon 3)CR003366 AAAUAGACACCAAAUCUUAC 36 gRNA targeting Human TTR (Exon 3)CR003367 AGACACCAAAUCUUACUGGA 37 gRNA targeting Human TTR (Exon 3)CR003368 AAGUGCCUUCCAGUAAGAUU 38 gRNA targeting Human TTR (Exon 3)CR003369 CUCUGCAUGCUCAUGGAAUG 39 gRNA targeting Human TTR (Exon 3)CR003370 CCUCUGCAUGCUCAUGGAAU 40 gRNA targeting Human TTR (Exon 3)CR003371 ACCUCUGCAUGCUCAUGGAA 41 gRNA targeting Human TTR (Exon 3)CR003372 UACUCACCUCUGCAUGCUCA 42 gRNA targeting Human TTR (Exon 3)CR003373 GUAUUCACAGCCAACGACUC 43 gRNA targeting Human TTR (Exon 4)CR003374 GCGGCGGGGGCCGGAGUCGU 44 gRNA targeting Human TTR (Exon 4)CR003375 AAUGGUGUAGCGGCGGGGGC 45 gRNA targeting Human TTR (Exon 4)CR003376 CGGCAAUGGUGUAGCGGCGG 46 gRNA targeting Human TTR (Exon 4)CR003377 GCGGCAAUGGUGUAGCGGCG 47 gRNA targeting Human TTR (Exon 4)CR003378 GGCGGCAAUGGUGUAGCGGC 48 gRNA targeting Human TTR (Exon 4)CR003379 GGGCGGCAAUGGUGUAGCGG 49 gRNA targeting Human TTR (Exon 4)CR003380 GCAGGGCGGCAAUGGUGUAG 50 gRNA targeting Human TTR (Exon 4)CR003381 GGGGCUCAGCAGGGCGGCAA 51 gRNA targeting Human TTR (Exon 4)CR003382 GGAGUAGGGGCUCAGCAGGG 52 gRNA targeting Human TTR (Exon 4)CR003383 AUAGGAGUAGGGGCUCAGCA 53 gRNA targeting Human TTR (Exon 4)CR003384 AAUAGGAGUAGGGGCUCAGC 54 gRNA targeting Human TTR (Exon 4)CR003385 CCCCUACUCCUAUUCCACCA 55 gRNA targeting Human TTR (Exon 4)CR003386 CCGUGGUGGAAUAGGAGUAG 56 gRNA targeting Human TTR (Exon 4)CR003387 GCCGUGGUGGAAUAGGAGUA 57 gRNA targeting Human TTR (Exon 4)CR003388 GACGACAGCCGUGGUGGAAU 58 gRNA targeting Human TTR (Exon 4)CR003389 AUUGGUGACGACAGCCGUGG 59 gRNA targeting Human TTR (Exon 4)CR003390 GGGAUUGGUGACGACAGCCG 60 gRNA targeting Human TTR (Exon 4)CR003391 GGCUGUCGUCACCAAUCCCA 61 gRNA targeting Human TTR (Exon 4)CR003392 AGUCCCUCAUUCCUUGGGAU 62 gRNA targeting Human TTR (Exon 4)CR005298 UCCACUCAUUCUUGGCAGGA 63 gRNA targeting Human TTR (Exon 1)CR005299 AGCCGUGGUGGAAUAGGAGU 64 gRNA targeting Human TTR (Exon 4)CR005300 UCACAGAAACACUCACCGUA 65 gRNA targeting Human TTR (Exon 1)CR005301 GUCACAGAAACACUCACCGU 66 gRNA targeting Human TTR (Exon 1)CR005302 ACGUGUCUUCUCUACACCCA 67 gRNA targeting Human TTR (Exon 2)CR005303 UGAAUCCAAGUGUCCUCUGA 68 gRNA targeting Human TTR (Exon 2)CR005304 GGCCGUGCAUGUGUUCAGAA 69 gRNA targeting Human TTR (Exon 2)CR005305 UAUAGGAAAACCAGUGAGUC 70 gRNA targeting Human TTR (Exon 3)CR005306 AAAUCUUACUGGAAGGCACU 71 gRNA targeting Human TTR (Exon 3)CR005307 UGUCUGUCUUCUCUCAUAGG 72 gRNA targeting Human TTR (Exon 4)CR000689 ACACAAAUACCAGUCCAGCG 73 gRNA targeting Cyno TTR CR005364AAAGGCUGCUGAUGAGACCU 74 gRNA targeting Cyno TTR CR005365CAUUGACAGCAGGACUGCCU 75 gRNA targeting Cyno TTR CR005366AUACCAGUCCAGCGAGGCAG 76 gRNA targeting Cyno TTR CR005367CCAGUCCAGCGAGGCAGAGG 77 gRNA targeting Cyno TTR CR005368CCUCCUCUGCCUCGCUGGAC 78 gRNA targeting Cyno TTR CR005369AAAGUUCUAGAUGCCGUCCG 79 gRNA targeting Cyno TTR CR005370ACUUGUCUUCUCUAUACCCA 80 gRNA targeting Cyno TTR CR005371AAGUGACUUCCAGUAAGAUU 81 gRNA targeting Cyno TTR CR005372AAAAGGCUGCUGAUGAGACC 82 gRNA targeting Cyno TTR Not Used 83 Not Used 84Not Used 85 Not Used 86 G000480mA*mA*mA*GGCUGCUGAUGACACCUGUUUUAGAmGmCmUmAmGmAmAmAm 87 sgRNAUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modifiedmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targetingHuman TTR G000481 mU*mC*mU*AGAACUUUGACCAUCAGGUUUUAGAmGmCmUmAmGmAmAmAm 88sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modifiedmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targetingHuman TTR G000482 mU*mG*mU*AGAAGGGAUAUACAAAGGUUUUAGAmGmCmUmAmGmAmAmAm 89sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modifiedmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targetingHuman TTR G000483 mU*mC*mC*ACUCAUUCUUGGCAGGAGUUUUAGAmGmCmUmAmGmAmAmAm 90sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modifiedmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targetingHuman TTR G000484 mA*mG*mA*CACCAAAUCUUACUGGAGUUUUAGAmGmCmUmAmGmAmAmAm 91sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modifiedmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targetingHuman TTR G000485 mC*mC*mU*CCUCUGCCUUGCUGGACGUUUUAGAmGmCmUmAmGmAmAmAm 92sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modifiedmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targetingHuman TTR G000486 mA*mC*mA*CAAAUACCAGUCCAGCAGUUUUAGAmGmCmUmAmGmAmAmAm 93sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modifiedmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targetingHuman TTR G000487 mU*mU*mC*UUUGGCAACUUACCCAGGUUUUAGAmGmCmUmAmGmAmAmAm 94sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modifiedmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targetingHuman TTR G000488 mA*mA*mA*GUUCUAGAUGCUGUCCGGUUUUAGAmGmCmUmAmGmAmAmAm 95sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modifiedmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targetingHuman TTR G000489 mU*mU*mU*GACCAUCAGAGGACACUGUUUUAGAmGmCmUmAmGmAmAmAm 96sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modifiedmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targetingHuman TTR G000490 mA*mA*mA*UAGACACCAAAUCUUACGUUUUAGAmGmCmUmAmGmAmAmAm 97sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modifiedmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targetingHuman TTR G000491 mA*mU*mA*CCAGUCCAGCAAGGCAGGUUUUAGAmGmCmUmAmGmAmAmAm 98sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modifiedmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targetingHuman TTR G000492 mC*mU*mU*CUCUACACCCAGGGCACGUUUUAGAmGmCmUmAmGmAmAmAm 99sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modifiedmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targetingHuman TTR G000493 mA*mA*mG*UGCCUUCCAGUAAGAUUGUUUUAGAmGmCmUmAmGmAmAmAm100 sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modifiedmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targetingHuman TTR G000494 mG*mU*mG*AGUCUGGAGAGCUGCAUGUUUUAGAmGmCmUmAmGmAmAmAm101 sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modifiedmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targetingHuman TTR G000495 mC*mA*mG*AGGACACUUGGAUUCACGUUUUAGAmGmCmUmAmGmAmAmAm102 sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modifiedmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targetingHuman TTR G000496 mG*mG*mC*CGUGCAUGUGUUCAGAAGUUUUAGAmGmCmUmAmGmAmAmAm103 sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modifiedmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targetingHuman TTR G000497 mC*mU*mG*CUCCUCCUCUGCCUUGCGUUUUAGAmGmCmUmAmGmAmAmAm104 sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modifiedmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targetingHuman TTR G000498 mA*mG*mU*GAGUCUGGAGAGCUGCAGUUUUAGAmGmCmUmAmGmAmAmAm105 sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modifiedmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targetingHuman TTR G000499 mU*mG*mA*AUCCAAGUGUCCUCUGAGUUUUAGAmGmCmUmAmGmAmAmAm106 sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modifiedmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targetingHuman TTR G000500 mC*mC*mA*GUCCAGCAAGGCAGAGGGUUUUAGAmGmCmUmAmGmAmAmAm107 sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modifiedmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targetingHuman TTR G000501 mU*mC*mA*CAGAAACACUCACCGUAGUUUUAGAmGmCmUmAmGmAmAmAm108 sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modifiedmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targetingHuman TTR G000567 mG*mA*mA*AGGCUGCUGAUGACACCGUUUUAGAmGmCmUmAmGmAmAmAm109 sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modifiedmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targetingHuman TTR G000568 mG*mG*mC*UGUCGUCACCAAUCCCAGUUUUAGAmGmCmUmAmGmAmAmAm110 sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modifiedmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targetingHuman TTR G000570 mC*mA*mU*UGAUGGCAGGACUGCCUGUUUUAGAmGmCmUmAmGmAmAmAm111 sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modifiedmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targetingHuman TTR G000571 mG*mU*mC*ACAGAAACACUCACCGUGUUUUAGAmGmCmUmAmGmAmAmAm112 sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modifiedmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targetingHuman TTR G000572 mC*mC*mC*CUACUCCUAUUCCACCAGUUUUAGAmGmCmUmAmGmAmAmAm113 sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modifiedmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targetingHuman TTR G000502 mA*mC*mA*CAAAUACCAGUCCAGCGGUUUUAGAmGmCmUmAmGmAmAmAm114 sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modifiedmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targetingCyno TTR G000503 mA*mA*mA*AGGCUGCUGAUGAGACCGUUUUAGAmGmCmUmAmGmAmAmAm 115sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modifiedmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targetingCyno TTR G000504 mA*mA*mA*GGCUGCUGAUGAGACCUGUUUUAGAmGmCmUmAmGmAmAmAm 116sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modifiedmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targetingCyno TTR G000505 mC*mA*mU*UGACAGCAGGACUGCCUGUUUUAGAmGmCmUmAmGmAmAmAm 117sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modifiedmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targetingCyno TTR G000506 mA*mU*mA*CCAGUCCAGCGAGGCAGGUUUUAGAmGmCmUmAmGmAmAmAm 118sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modifiedmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targetingCyno TTR G000507 mC*mC*mA*GUCCAGCGAGGCAGAGGGUUUUAGAmGmCmUmAmGmAmAmAm 119sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modifiedmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targetingCyno TTR G000508 mC*mC*mU*CCUCUGCCUCGCUGGACGUUUUAGAmGmCmUmAmGmAmAmAm 120sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modifiedmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targetingCyno TTR G000509 mA*mA*mA*GUUCUAGAUGCCGUCCGGUUUUAGAmGmCmUmAmGmAmAmAm 121sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modifiedmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targetingCyno TTR G000510 mA*mC*mU*UGUCUUCUCUAUACCCAGUUUUAGAmGmCmUmAmGmAmAmAm 122sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modifiedmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targetingCyno TTR G000511 mA*mA*mG*UGACUUCCAGUAAGAUUGUUUUAGAmGmCmUmAmGmAmAmAm 123sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modifiedmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targetingCyno TTR G000282 mU*mU*mA*CAGCCACGUCUACAGCAGUUUUAGAmGmCmUmAmGmAmAmAm 124sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modifiedmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targetingMouse TTR Not used 125 to 200 DNA codingATGGACAAGAAGTACAGCATCGGACTGGACATCGGAACAAACAGCGTCGGA 201 sequence ofTGGGCAGTCATCACAGACGAATACAAGGTCCCGAGCAAGAAGTTCAAGGTC Cas9 usingCTGGGAAACACAGACAGACACAGCATCAAGAAGAACCTGATCGGAGCACTG theCTGTTCGACAGCGGAGAAACAGCAGAAGCAACAAGACTGAAGAGAACAGCA thymidineAGAAGAAGATACACAAGAAGAAAGAACAGAATCTGCTACCTGCAGGAAATC analog ofTTCAGCAACGAAATGGCAAAGGTCGACGACAGCTTCTTCCACAGACTGGAA the minimalGAAAGCTTCCTGGTCGAAGAAGACAAGAAGCACGAAAGACACCCGATCTTC uridineGGAAACATCGTCGACGAAGTCGCATACCACGAAAAGTACCCGACAATCTAC codonsCACCTGAGAAAGAAGCTGGTCGACAGCACAGACAAGGCAGACCTGAGACTG listed inATCTACCTGGCACTGGCACACATGATCAAGTTCAGAGGACACTTCCTGATC Table 3,GAAGGAGACCTGAACCCGGACAACAGCGACGTCGACAAGCTGTTCATCCAG with startCTGGTCCAGACATACAACCAGCTGTTCGAAGAAAACCCGATCAACGCAAGC and stopGGAGTCGACGCAAAGGCAATCCTGAGCGCAAGACTGAGCAAGAGCAGAAGA codonsCTGGAAAACCTGATCGCACAGCTGCCGGGAGAAAAGAAGAACGGACTGTTCGGAAACCTGATCGCACTGAGCCTGGGACTGACACCGAACTTCAAGAGCAACTTCGACCTGGCAGAAGACGCAAAGCTGCAGCTGAGCAAGGACACATACGACGACGACCTGGACAACCTGCTGGCACAGATCGGAGACCAGTACGCAGACCTGTTCCTGGCAGCAAAGAACCTGAGCGACGCAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAAATCACAAAGGCACCGCTGAGCGCAAGCATGATCAAGAGATACGACGAACACCACCAGGACCTGACACTGCTGAAGGCACTGGTCAGACAGCAGCTGCCGGAAAAGTACAAGGAAATCTTCTTCGACCAGAGCAAGAACGGATACGCAGGATACATCGACGGAGGAGCAAGCCAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAAGATGGACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAAGACCTGCTGAGAAAGCAGAGAACATTCGACAACGGAAGCATCCCGCACCAGATCCACCTGGGAGAACTGCACGCAATCCTGAGAAGACAGGAAGACTTCTACCCGTTCCTGAAGGACAACAGAGAAAAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTACGTCGGACCGCTGGCAAGAGGAAACAGCAGATTCGCATGGATGACAAGAAAGAGCGAAGAAACAATCACACCGTGGAACTTCGAAGAAGTCGTCGACAAGGGAGCAAGCGCACAGAGCTTCATCGAAAGAATGACAAACTTCGACAAGAACCTGCCGAACGAAAAGGTCCTGCCGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTACAACGAACTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCCGGCATTCCTGAGCGGAGAACAGAAGAAGGCAATCGTCGACCTGCTGTTCAAGACAAACAGAAAGGTCACAGTCAAGCAGCTGAAGGAAGACTACTTCAAGAAGATCGAATGCTTCGACAGCGTCGAAATCAGCGGAGTCGAAGACAGATTCAACGCAAGCCTGGGAACATACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGACAACGAAGAAAACGAAGACATCCTGGAAGACATCGTCCTGACACTGACACTGTTCGAAGACAGAGAAATGATCGAAGAAAGACTGAAGACATACGCACACCTGTTCGACGACAAGGTCATGAAGCAGCTGAAGAGAAGAAGATACACAGGATGGGGAAGACTGAGCAGAAAGCTGATCAACGGAATCAGAGACAAGCAGAGCGGAAAGACAATCCTGGACTTCCTGAAGAGCGACGGATTCGCAAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACATTCAAGGAAGACATCCAGAAGGCACAGGTCAGCGGACAGGGAGACAGCCTGCACGAACACATCGCAAACCTGGCAGGAAGCCCGGCAATCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAACTGGTCAAGGTCATGGGAAGACACAAGCCGGAAAACATCGTCATCGAAATGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAATGAAGAGAATCGAAGAAGGAATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACACCCGGTCGAAAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCTGCAGAACGGAAGAGACATGTACGTCGACCAGGAACTGGACATCAACAGACTGAGCGACTACGACGTCGACCACATCGTCCCGCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTCCTGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGTCCCGAGCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGACAGCTGCTGAACGCAAAGCTGATCACACAGAGAAAGTTCGACAACCTGACAAAGGCAGAGAGAGGAGGACTGAGCGAACTGGACAAGGCAGGATTCATCAAGAGACAGCTGGTCGAAACAAGACAGATCACAAAGCACGTCGCACAGATCCTGGACAGCAGAATGAACACAAAGTACGACGAAAACGACAAGCTGATCAGAGAAGTCAAGGTCATCACACTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGACTTCCAGTTCTACAAGGTCAGAGAAATCAACAACTACCACCACGCACACGACGCATACCTGAACGCAGTCGTCGGAACAGCACTGATCAAGAAGTACCCGAAGCTGGAAAGCGAATTCGTCTACGGAGACTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGAGCGAACAGGAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAAGACAGAAATCACACTGGCAAACGGAGAAATCAGAAAGAGACCGCTGATCGAAACAAACGGAGAAACAGGAGAAATCGTCTGGGACAAGGGAAGAGACTTCGCAACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCGTCAAGAAGACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAGCATCCTGCCGAAGAGAAACAGCGACAAGCTGATCGCAAGAAAGAAGGACTGGGACCCGAAGAAGTACGGAGGATTCGACAGCCCGACAGTCGCATACAGCGTCCTGGTCGTCGCAAAGGTCGAAAAGGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGCTGGGAATCACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGATCGACTTCCTGGAAGCAAAGGGATACAAGGAAGTCAAGAAGGACCTGATCATCAAGCTGCCGAAGTACAGCCTGTTCGAACTGGAAAACGGAAGAAAGAGAATGCTGGCAAGCGCAGGAGAACTGCAGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGTCAACTTCCTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCTGTTCGTCGAACAGCACAAGCACTACCTGGACGAAATCATCGAACAGATCAGCGAATTCAGCAAGAGAGTCATCCTGGCAGACGCAAACCTGGACAAGGTCCTGAGCGCATACAACAAGCACAGAGACAAGCCGATCAGAGAACAGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTGGGAGCACCGGCAGCATTCAAGTACTTCGACACAACAATCGACAGAAAGAGATACACAAGCACAAAGGAAGTCCTGGACGCAACACTGATCCACCAGAGCATCACAGGACTGTACGAAACAAGAATCGACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAAGCCCGAAGAAGAAGAGA AAGGTCTAG DNA codingATGGATAAGAAGTACTCAATCGGGCTGGATATCGGAACTAATTCCGTGGGT 202 sequence ofTGGGCAGTGATCACGGATGAATACAAAGTGCCGTCCAAGAAGTTCAAGGTC Cas9 usingCTGGGGAACACCGATAGACACAGCATCAAGAAAAATCTCATCGGAGCCCTG codons withCTGTTTGACTCCGGCGAAACCGCAGAAGCGACCCGGCTCAAACGTACCGCG generallyAGGCGACGCTACACCCGGCGGAAGAATCGCATCTGCTATCTGCAAGAGATC highTTTTCGAACGAAATGGCAAAGGTCGACGACAGCTTCTTCCACCGCCTGGAA expressionGAATCTTTCCTGGTGGAGGAGGACAAGAAGCATGAACGGCATCCTATCTTT in humansGGAAACATCGTCGACGAAGTGGCGTACCACGAAAAGTACCCGACCATCTACCATCTGCGGAAGAAGTTGGTTGACTCAACTGACAAGGCCGACCTCAGATTGATCTACTTGGCCCTCGCCCATATGATCAAATTCCGCGGACACTTCCTGATCGAAGGCGATCTGAACCCTGATAACTCCGACGTGGATAAGCTTTTCATTCAACTGGTGCAGACCTACAACCAACTGTTCGAAGAAAACCCAATCAATGCTAGCGGCGTCGATGCCAAGGCCATCCTGTCCGCCCGGCTGTCGAAGTCGCGGCGCCTCGAAAACCTGATCGCACAGCTGCCGGGAGAGAAAAAGAACGGACTTTTCGGCAACTTGATCGCTCTCTCACTGGGACTCACTCCCAATTTCAAGTCCAATTTTGACCTGGCCGAGGACGCGAAGCTGCAACTCTCAAAGGACACCTACGACGACGACTTGGACAATTTGCTGGCACAAATTGGCGATCAGTACGCGGATCTGTTCCTTGCCGCTAAGAACCTTTCGGACGCAATCTTGCTGTCCGATATCCTGCGCGTGAACACCGAAATAACCAAAGCGCCGCTTAGCGCCTCGATGATTAAGCGGTACGACGAGCATCACCAGGATCTCACGCTGCTCAAAGCGCTCGTGAGACAGCAACTGCCTGAAAAGTACAAGGAGATCTTCTTCGACCAGTCCAAGAATGGGTACGCAGGGTACATCGATGGAGGCGCTAGCCAGGAAGAGTTCTATAAGTTCATCAAGCCAATCCTGGAAAAGATGGACGGAACCGAAGAACTGCTGGTCAAGCTGAACAGGGAGGATCTGCTCCGGAAACAGAGAACCTTTGACAACGGATCCATTCCCCACCAGATCCATCTGGGTGAGCTGCACGCCATCTTGCGGCGCCAGGAGGACTTTTACCCATTCCTCAAGGACAACCGGGAAAAGATCGAGAAAATTCTGACGTTCCGCATCCCGTATTACGTGGGCCCACTGGCGCGCGGCAATTCGCGCTTCGCGTGGATGACTAGAAAATCAGAGGAAACCATCACTCCTTGGAATTTCGAGGAAGTTGTGGATAAGGGAGCTTCGGCACAAAGCTTCATCGAACGAATGACCAACTTCGACAAGAATCTCCCAAACGAGAAGGTGCTTCCTAAGCACAGCCTCCTTTACGAATACTTCACTGTCTACAACGAACTGACTAAAGTGAAATACGTTACTGAAGGAATGAGGAAGCCGGCCTTTCTGTCCGGAGAACAGAAGAAAGCAATTGTCGATCTGCTGTTCAAGACCAACCGCAAGGTGACCGTCAAGCAGCTTAAAGAGGACTACTTCAAGAAGATCGAGTGTTTCGACTCAGTGGAAATCAGCGGGGTGGAGGACAGATTCAACGCTTCGCTGGGAACCTATCATGATCTCCTGAAGATCATCAAGGACAAGGACTTCCTTGACAACGAGGAGAACGAGGACATCCTGGAAGATATCGTCCTGACCTTGACCCTTTTCGAGGATCGCGAGATGATCGAGGAGAGGCTTAAGACCTACGCTCATCTCTTCGACGATAAGGTCATGAAACAACTCAAGCGCCGCCGGTACACTGGTTGGGGCCGCCTCTCCCGCAAGCTGATCAACGGTATTCGCGATAAACAGAGCGGTAAAACTATCCTGGATTTCCTCAAATCGGATGGCTTCGCTAATCGTAACTTCATGCAATTGATCCACGACGACAGCCTGACCTTTAAGGAGGACATCCAAAAAGCACAAGTGTCCGGACAGGGAGACTCACTCCATGAACACATCGCGAATCTGGCCGGTTCGCCGGCGATTAAGAAGGGAATTCTGCAAACTGTGAAGGTGGTCGACGAGCTGGTGAAGGTCATGGGACGGCACAAACCGGAGAATATCGTGATTGAAATGGCCCGAGAAAACCAGACTACCCAGAAGGGCCAGAAAAACTCCCGCGAAAGGATGAAGCGGATCGAAGAAGGAATCAAGGAGCTGGGCAGCCAGATCCTGAAAGAGCACCCGGTGGAAAACACGCAGCTGCAGAACGAGAAGCTCTACCTGTACTATTTGCAAAATGGACGGGACATGTACGTGGACCAAGAGCTGGACATCAATCGGTTGTCTGATTACGACGTGGACCACATCGTTCCACAGTCCTTTCTGAAGGATGACTCGATCGATAACAAGGTGTTGACTCGCAGCGACAAGAACAGAGGGAAGTCAGATAATGTGCCATCGGAGGAGGTCGTGAAGAAGATGAAGAATTACTGGCGGCAGCTCCTGAATGCGAAGCTGATTACCCAGAGAAAGTTTGACAATCTCACTAAAGCCGAGCGCGGCGGACTCTCAGAGCTGGATAAGGCTGGATTCATCAAACGGCAGCTGGTCGAGACTCGGCAGATTACCAAGCACGTGGCGCAGATCTTGGACTCCCGCATGAACACTAAATACGACGAGAACGATAAGCTCATCCGGGAAGTGAAGGTGATTACCCTGAAAAGCAAACTTGTGTCGGACTTTCGGAAGGACTTTCAGTTTTACAAAGTGAGAGAAATCAACAACTACCATCACGCGCATGACGCATACCTCAACGCTGTGGTCGGTACCGCCCTGATCAAAAAGTACCCTAAACTTGAATCGGAGTTTGTGTACGGAGACTACAAGGTCTACGACGTGAGGAAGATGATAGCCAAGTCCGAACAGGAAATCGGGAAAGCAACTGCGAAATACTTCTTTTACTCAAACATCATGAACTTTTTCAAGACTGAAATTACGCTGGCCAATGGAGAAATCAGGAAGAGGCCACTGATCGAAACTAACGGAGAAACGGGCGAAATCGTGTGGGACAAGGGCAGGGACTTCGCAACTGTTCGCAAAGTGCTCTCTATGCCGCAAGTCAATATTGTGAAGAAAACCGAAGTGCAAACCGGCGGATTTTCAAAGGAATCGATCCTCCCAAAGAGAAATAGCGACAAGCTCATTGCACGCAAGAAAGACTGGGACCCGAAGAAGTACGGAGGATTCGATTCGCCGACTGTCGCATACTCCGTCCTCGTGGTGGCCAAGGTGGAGAAGGGAAAGAGCAAAAAGCTCAAATCCGTCAAAGAGCTGCTGGGGATTACCATCATGGAACGATCCTCGTTCGAGAAGAACCCGATTGATTTCCTCGAGGCGAAGGGTTACAAGGAGGTGAAGAAGGATCTGATCATCAAACTCCCCAAGTACTCACTGTTCGAACTGGAAAATGGTCGGAAGCGCATGCTGGCTTCGGCCGGAGAACTCCAAAAAGGAAATGAGCTGGCCTTGCCTAGCAAGTACGTCAACTTCCTCTATCTTGCTTCGCACTACGAAAAACTCAAAGGGTCACCGGAAGATAACGAACAGAAGCAGCTTTTCGTGGAGCAGCACAAGCATTATCTGGATGAAATCATCGAACAAATCTCCGAGTTTTCAAAGCGCGTGATCCTCGCCGACGCCAACCTCGACAAAGTCCTGTCGGCCTACAATAAGCATAGAGATAAGCCGATCAGAGAACAGGCCGAGAACATTATCCACTTGTTCACCCTGACTAACCTGGGAGCCCCAGCCGCCTTCAAGTACTTCGATACTACTATCGATCGCAAAAGATACACGTCCACCAAGGAAGTTCTGGACGCGACCCTGATCCACCAAAGCATCACTGGACTCTACGAAACTAGGATCGATCTGTCGCAGCTGGGTGGCGATGGCGGTGGATCTCCGAAAAAGAAGAGA AAGGTGTAATGAAmino acid MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGAL 203sequence of LFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLECas9 with ESELVEEDKKHERHPIEGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLone nuclear IYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASlocalization GVDAKAILSARLSKSRRLENLIAQLPGEKKNGLEGNLIALSLGLTPNEKSN signalFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDIL (1xNLS) asRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKN the C-GYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNG terminal 7SIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN amino acidsSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAELSGEQKKAIVDLLEKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTEKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKEDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFEKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGESKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENITHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDGGGSPKKKR KV Cas9 mRNAAUGGACAAGAAGUACAGCAUCGGACUGGACAUCGGAACAAACAGCGUCGGA 204 ORF usingUGGGCAGUCAUCACAGACGAAUACAAGGUCCCGAGCAAGAAGUUCAAGGUC minimalCUGGGAAACACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGAGCACUG uridineCUGUUCGACAGCGGAGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCA codons, withAGAAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAGGAAAUC start andUUCAGCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCACAGACUGGAA stop codonsGAAAGCUUCCUGGUCGAAGAAGACAAGAAGCACGAAAGACACCCGAUCUUCGGAAACAUCGUCGACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUACCACCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACCUGAGACUGAUCUACCUGGCACUGGCACACAUGAUCAAGUUCAGAGGACACUUCCUGAUCGAAGGAGACCUGAACCCGGACAACAGCGACGUCGACAAGCUGUUCAUCCAGCUGGUCCAGACAUACAACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGAGUCGACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAGCAGAAGACUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAAAGAAGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUGGGACUGACACCGAACUUCAAGAGCAACUUCGACCUGGCAGAAGACGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGACGACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCAGACCUGUUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCUGCUGAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGGCACCGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACACCACCAGGACCUGACACUGCUGAAGGCACUGGUCAGACAGCAGCUGCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCAAGAACGGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAGGAAGAAUUCUACAAGUUCAUCAAGCCGAUCCUGGAAAAGAUGGACGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAGACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCCCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAGACAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGAAAAGAUCGAAAAGAUCCUGACAUUCAGAAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAACAGCAGAUUCGCAUGGAUGACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUCGAAGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAAAGAAUGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAAGCACAGCCUGCUGUACGAAUACUUCACAGUCUACAACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAAUGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCAAUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGUCAAGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUCGACAGCGUCGAAAUCAGCGGAGUCGAAGACAGAUUCAACGCAAGCCUGGGAACAUACCACGACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAAGAAAACGAAGACAUCCUGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGAGAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCGACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUCAGAGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCCACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCACAGGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGCAAACCUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGCAGACAGUCAAGGUCGUCGACGAACUGGUCAAGGUCAUGGGAAGACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAAUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGCCAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAGAACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAAGAGACAUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGCGACUACGACGUCGACCACAUCGUCCCGCAGAGCUUCCUGAAGGACGACAGCAUCGACAACAAGGUCCUGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGUCGUCAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCAAAGCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAGGCAGAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAUCAAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAGCACGUCGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUACGACGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACACUGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAGUUCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACACGACGCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAGCUGGAAAGCGAAUUCGUCUACGGAGACUACAAGGUCUACGACGUCAGAAAGAUGAUCGCAAAGAGCGAACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACAGCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAACGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGGAGAAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGACUUCGCAACAGUCAGAAAGGUCCUGAGCAUGCCGCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGCAAGGAAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGAAAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAGUCGCAUACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAACUGCUGGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUCAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUCGAACUGGAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCAGGAGAACUGCAGAAGGGAAACGAACUGGCACUGCCGAGCAAGUACGUCAACUUCCUGUACCUGGCAAGCCACUACGAAAAGCUGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUCGUCGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACAGAUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAAACCUGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACAAGCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUGUUCACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUACUUCGACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAGGAAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACAGGACUGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAGACGGAGGAGGAAGCCCGAAGAAGAAGAGA AAGGUCUAG Cas9 mRNAAUGGAUAAGAAGUACUCAAUCGGGCUGGAUAUCGGAACUAAUUCCGUGGGU 205 ORF usingUGGGCAGUGAUCACGGAUGAAUACAAAGUGCCGUCCAAGAAGUUCAAGGUC codons withCUGGGGAACACCGAUAGACACAGCAUCAAGAAAAAUCUCAUCGGAGCCCUG generallyCUGUUUGACUCCGGCGAAACCGCAGAAGCGACCCGGCUCAAACGUACCGCG highAGGCGACGCUACACCCGGCGGAAGAAUCGCAUCUGCUAUCUGCAAGAGAUC expressionUUUUCGAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCACCGCCUGGAA in humans,GAAUCUUUCCUGGUGGAGGAGGACAAGAAGCAUGAACGGCAUCCUAUCUUU with startGGAAACAUCGUCGACGAAGUGGCGUACCACGAAAAGUACCCGACCAUCUAC and stopCAUCUGCGGAAGAAGUUGGUUGACUCAACUGACAAGGCCGACCUCAGAUUG codonsAUCUACUUGGCCCUCGCCCAUAUGAUCAAAUUCCGCGGACACUUCCUGAUCGAAGGCGAUCUGAACCCUGAUAACUCCGACGUGGAUAAGCUUUUCAUUCAACUGGUGCAGACCUACAACCAACUGUUCGAAGAAAACCCAAUCAAUGCUAGCGGCGUCGAUGCCAAGGCCAUCCUGUCCGCCCGGCUGUCGAAGUCGCGGCGCCUCGAAAACCUGAUCGCACAGCUGCCGGGAGAGAAAAAGAACGGACUUUUCGGCAACUUGAUCGCUCUCUCACUGGGACUCACUCCCAAUUUCAAGUCCAAUUUUGACCUGGCCGAGGACGCGAAGCUGCAACUCUCAAAGGACACCUACGACGACGACUUGGACAAUUUGCUGGCACAAAUUGGCGAUCAGUACGCGGAUCUGUUCCUUGCCGCUAAGAACCUUUCGGACGCAAUCUUGCUGUCCGAUAUCCUGCGCGUGAACACCGAAAUAACCAAAGCGCCGCUUAGCGCCUCGAUGAUUAAGCGGUACGACGAGCAUCACCAGGAUCUCACGCUGCUCAAAGCGCUCGUGAGACAGCAACUGCCUGAAAAGUACAAGGAGAUCUUCUUCGACCAGUCCAAGAAUGGGUACGCAGGGUACAUCGAUGGAGGCGCUAGCCAGGAAGAGUUCUAUAAGUUCAUCAAGCCAAUCCUGGAAAAGAUGGACGGAACCGAAGAACUGCUGGUCAAGCUGAACAGGGAGGAUCUGCUCCGGAAACAGAGAACCUUUGACAACGGAUCCAUUCCCCACCAGAUCCAUCUGGGUGAGCUGCACGCCAUCUUGCGGCGCCAGGAGGACUUUUACCCAUUCCUCAAGGACAACCGGGAAAAGAUCGAGAAAAUUCUGACGUUCCGCAUCCCGUAUUACGUGGGCCCACUGGCGCGCGGCAAUUCGCGCUUCGCGUGGAUGACUAGAAAAUCAGAGGAAACCAUCACUCCUUGGAAUUUCGAGGAAGUUGUGGAUAAGGGAGCUUCGGCACAAAGCUUCAUCGAACGAAUGACCAACUUCGACAAGAAUCUCCCAAACGAGAAGGUGCUUCCUAAGCACAGCCUCCUUUACGAAUACUUCACUGUCUACAACGAACUGACUAAAGUGAAAUACGUUACUGAAGGAAUGAGGAAGCCGGCCUUUCUGUCCGGAGAACAGAAGAAAGCAAUUGUCGAUCUGCUGUUCAAGACCAACCGCAAGGUGACCGUCAAGCAGCUUAAAGAGGACUACUUCAAGAAGAUCGAGUGUUUCGACUCAGUGGAAAUCAGCGGGGUGGAGGACAGAUUCAACGCUUCGCUGGGAACCUAUCAUGAUCUCCUGAAGAUCAUCAAGGACAAGGACUUCCUUGACAACGAGGAGAACGAGGACAUCCUGGAAGAUAUCGUCCUGACCUUGACCCUUUUCGAGGAUCGCGAGAUGAUCGAGGAGAGGCUUAAGACCUACGCUCAUCUCUUCGACGAUAAGGUCAUGAAACAACUCAAGCGCCGCCGGUACACUGGUUGGGGCCGCCUCUCCCGCAAGCUGAUCAACGGUAUUCGCGAUAAACAGAGCGGUAAAACUAUCCUGGAUUUCCUCAAAUCGGAUGGCUUCGCUAAUCGUAACUUCAUGCAAUUGAUCCACGACGACAGCCUGACCUUUAAGGAGGACAUCCAAAAAGCACAAGUGUCCGGACAGGGAGACUCACUCCAUGAACACAUCGCGAAUCUGGCCGGUUCGCCGGCGAUUAAGAAGGGAAUUCUGCAAACUGUGAAGGUGGUCGACGAGCUGGUGAAGGUCAUGGGACGGCACAAACCGGAGAAUAUCGUGAUUGAAAUGGCCCGAGAAAACCAGACUACCCAGAAGGGCCAGAAAAACUCCCGCGAAAGGAUGAAGCGGAUCGAAGAAGGAAUCAAGGAGCUGGGCAGCCAGAUCCUGAAAGAGCACCCGGUGGAAAACACGCAGCUGCAGAACGAGAAGCUCUACCUGUACUAUUUGCAAAAUGGACGGGACAUGUACGUGGACCAAGAGCUGGACAUCAAUCGGUUGUCUGAUUACGACGUGGACCACAUCGUUCCACAGUCCUUUCUGAAGGAUGACUCGAUCGAUAACAAGGUGUUGACUCGCAGCGACAAGAACAGAGGGAAGUCAGAUAAUGUGCCAUCGGAGGAGGUCGUGAAGAAGAUGAAGAAUUACUGGCGGCAGCUCCUGAAUGCGAAGCUGAUUACCCAGAGAAAGUUUGACAAUCUCACUAAAGCCGAGCGCGGCGGACUCUCAGAGCUGGAUAAGGCUGGAUUCAUCAAACGGCAGCUGGUCGAGACUCGGCAGAUUACCAAGCACGUGGCGCAGAUCUUGGACUCCCGCAUGAACACUAAAUACGACGAGAACGAUAAGCUCAUCCGGGAAGUGAAGGUGAUUACCCUGAAAAGCAAACUUGUGUCGGACUUUCGGAAGGACUUUCAGUUUUACAAAGUGAGAGAAAUCAACAACUACCAUCACGCGCAUGACGCAUACCUCAACGCUGUGGUCGGUACCGCCCUGAUCAAAAAGUACCCUAAACUUGAAUCGGAGUUUGUGUACGGAGACUACAAGGUCUACGACGUGAGGAAGAUGAUAGCCAAGUCCGAACAGGAAAUCGGGAAAGCAACUGCGAAAUACUUCUUUUACUCAAACAUCAUGAACUUUUUCAAGACUGAAAUUACGCUGGCCAAUGGAGAAAUCAGGAAGAGGCCACUGAUCGAAACUAACGGAGAAACGGGCGAAAUCGUGUGGGACAAGGGCAGGGACUUCGCAACUGUUCGCAAAGUGCUCUCUAUGCCGCAAGUCAAUAUUGUGAAGAAAACCGAAGUGCAAACCGGCGGAUUUUCAAAGGAAUCGAUCCUCCCAAAGAGAAAUAGCGACAAGCUCAUUGCACGCAAGAAAGACUGGGACCCGAAGAAGUACGGAGGAUUCGAUUCGCCGACUGUCGCAUACUCCGUCCUCGUGGUGGCCAAGGUGGAGAAGGGAAAGAGCAAAAAGCUCAAAUCCGUCAAAGAGCUGCUGGGGAUUACCAUCAUGGAACGAUCCUCGUUCGAGAAGAACCCGAUUGAUUUCCUCGAGGCGAAGGGUUACAAGGAGGUGAAGAAGGAUCUGAUCAUCAAACUCCCCAAGUACUCACUGUUCGAACUGGAAAAUGGUCGGAAGCGCAUGCUGGCUUCGGCCGGAGAACUCCAAAAAGGAAAUGAGCUGGCCUUGCCUAGCAAGUACGUCAACUUCCUCUAUCUUGCUUCGCACUACGAAAAACUCAAAGGGUCACCGGAAGAUAACGAACAGAAGCAGCUUUUCGUGGAGCAGCACAAGCAUUAUCUGGAUGAAAUCAUCGAACAAAUCUCCGAGUUUUCAAAGCGCGUGAUCCUCGCCGACGCCAACCUCGACAAAGUCCUGUCGGCCUACAAUAAGCAUAGAGAUAAGCCGAUCAGAGAACAGGCCGAGAACAUUAUCCACUUGUUCACCCUGACUAACCUGGGAGCCCCAGCCGCCUUCAAGUACUUCGAUACUACUAUCGAUCGCAAAAGAUACACGUCCACCAAGGAAGUUCUGGACGCGACCCUGAUCCACCAAAGCAUCACUGGACUCUACGAAACUAGGAUCGAUCUGUCGCAGCUGGGUGGCGAUGGCGGUGGAUCUCCGAAAAAGAAGAGA AAGGUGUAAUGACas9 nickase MDKKYSIGLAIGINSVGWAVITDEYKVPSKKFKVLGNIDRHSIKKNLIGAL 206(D10A) amino LFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLE acidESELVEEDKKHERHPIEGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRL sequenceIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLEGNLIALSLGLIPNEKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLILLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMINFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLEKTNRKVIVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLILTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLIFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLIRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKEDNLIKAERGGLSELDKAGFIKRQLVETRQIIKHVAQILDSRMNIKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGIALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFEKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGESKESILPKRNSDKLIARKKDWDPKKYGGFDSPIVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDGGGSPKKKR KV Cas9 nickaseAUGGACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAAACAGCGUCGGA 207 (D10A) mRNAUGGGCAGUCAUCACAGACGAAUACAAGGUCCCGAGCAAGAAGUUCAAGGUC ORFCUGGGAAACACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGAGCACUGCUGUUCGACAGCGGAGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCAAGAAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAGGAAAUCUUCAGCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCACAGACUGGAAGAAAGCUUCCUGGUCGAAGAAGACAAGAAGCACGAAAGACACCCGAUCUUCGGAAACAUCGUCGACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUACCACCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACCUGAGACUGAUCUACCUGGCACUGGCACACAUGAUCAAGUUCAGAGGACACUUCCUGAUCGAAGGAGACCUGAACCCGGACAACAGCGACGUCGACAAGCUGUUCAUCCAGCUGGUCCAGACAUACAACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGAGUCGACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAGCAGAAGACUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAAAGAAGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUGGGACUGACACCGAACUUCAAGAGCAACUUCGACCUGGCAGAAGACGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGACGACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCAGACCUGUUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCUGCUGAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGGCACCGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACACCACCAGGACCUGACACUGCUGAAGGCACUGGUCAGACAGCAGCUGCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCAAGAACGGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAGGAAGAAUUCUACAAGUUCAUCAAGCCGAUCCUGGAAAAGAUGGACGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAGACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCCCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAGACAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGAAAAGAUCGAAAAGAUCCUGACAUUCAGAAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAACAGCAGAUUCGCAUGGAUGACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUCGAAGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAAAGAAUGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAAGCACAGCCUGCUGUACGAAUACUUCACAGUCUACAACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAAUGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCAAUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGUCAAGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUCGACAGCGUCGAAAUCAGCGGAGUCGAAGACAGAUUCAACGCAAGCCUGGGAACAUACCACGACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAAGAAAACGAAGACAUCCUGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGAGAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCGACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUCAGAGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCCACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCACAGGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGCAAACCUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGCAGACAGUCAAGGUCGUCGACGAACUGGUCAAGGUCAUGGGAAGACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAAUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGCCAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAGAACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAAGAGACAUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGCGACUACGACGUCGACCACAUCGUCCCGCAGAGCUUCCUGAAGGACGACAGCAUCGACAACAAGGUCCUGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGUCGUCAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCAAAGCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAGGCAGAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAUCAAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAGCACGUCGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUACGACGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACACUGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAGUUCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACACGACGCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAGCUGGAAAGCGAAUUCGUCUACGGAGACUACAAGGUCUACGACGUCAGAAAGAUGAUCGCAAAGAGCGAACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACAGCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAACGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGGAGAAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGACUUCGCAACAGUCAGAAAGGUCCUGAGCAUGCCGCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGCAAGGAAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGAAAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAGUCGCAUACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAACUGCUGGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUCAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUCGAACUGGAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCAGGAGAACUGCAGAAGGGAAACGAACUGGCACUGCCGAGCAAGUACGUCAACUUCCUGUACCUGGCAAGCCACUACGAAAAGCUGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUCGUCGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACAGAUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAAACCUGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACAAGCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUGUUCACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUACUUCGACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAGGAAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACAGGACUGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAGACGGAGGAGGAAGCCCGAAGAAGAAGAGA AAGGUCUAGdCas9 (D10A MDKKYSIGLAIGINSVGWAVITDEYKVPSKKFKVLGNIDRHSIKKNLIGAL 208H840A) amino LFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLE acidESELVEEDKKHERHPIEGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRL sequenceIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLEGNLIALSLGLIPNEKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLILLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMINFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLEKTNRKVIVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLILTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLIFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLIRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKEDNLIKAERGGLSELDKAGFIKRQLVETRQIIKHVAQILDSRMNIKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGIALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFEKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGESKESILPKRNSDKLIARKKDWDPKKYGGFDSPIVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLINLGAPAAFKYFDTTIDRKRYISTKEVLDATLIHQSITGLYETRIDLSQLGGDGGGSPKKKR KV dCas9 (D10AAUGGACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAAACAGCGUCGGA 209 H840A) mRNAUGGGCAGUCAUCACAGACGAAUACAAGGUCCCGAGCAAGAAGUUCAAGGUC ORFCUGGGAAACACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGAGCACUGCUGUUCGACAGCGGAGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCAAGAAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAGGAAAUCUUCAGCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCACAGACUGGAAGAAAGCUUCCUGGUCGAAGAAGACAAGAAGCACGAAAGACACCCGAUCUUCGGAAACAUCGUCGACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUACCACCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACCUGAGACUGAUCUACCUGGCACUGGCACACAUGAUCAAGUUCAGAGGACACUUCCUGAUCGAAGGAGACCUGAACCCGGACAACAGCGACGUCGACAAGCUGUUCAUCCAGCUGGUCCAGACAUACAACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGAGUCGACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAGCAGAAGACUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAAAGAAGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUGGGACUGACACCGAACUUCAAGAGCAACUUCGACCUGGCAGAAGACGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGACGACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCAGACCUGUUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCUGCUGAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGGCACCGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACACCACCAGGACCUGACACUGCUGAAGGCACUGGUCAGACAGCAGCUGCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCAAGAACGGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAGGAAGAAUUCUACAAGUUCAUCAAGCCGAUCCUGGAAAAGAUGGACGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAGACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCCCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAGACAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGAAAAGAUCGAAAAGAUCCUGACAUUCAGAAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAACAGCAGAUUCGCAUGGAUGACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUCGAAGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAAAGAAUGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAAGCACAGCCUGCUGUACGAAUACUUCACAGUCUACAACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAAUGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCAAUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGUCAAGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUCGACAGCGUCGAAAUCAGCGGAGUCGAAGACAGAUUCAACGCAAGCCUGGGAACAUACCACGACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAAGAAAACGAAGACAUCCUGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGAGAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCGACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUCAGAGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCCACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCACAGGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGCAAACCUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGCAGACAGUCAAGGUCGUCGACGAACUGGUCAAGGUCAUGGGAAGACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAAUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGCCAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAGAACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAAGAGACAUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGCGACUACGACGUCGACGCAAUCGUCCCGCAGAGCUUCCUGAAGGACGACAGCAUCGACAACAAGGUCCUGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGUCGUCAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCAAAGCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAGGCAGAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAUCAAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAGCACGUCGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUACGACGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACACUGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAGUUCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACACGACGCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAGCUGGAAAGCGAAUUCGUCUACGGAGACUACAAGGUCUACGACGUCAGAAAGAUGAUCGCAAAGAGCGAACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACAGCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAACGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGGAGAAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGACUUCGCAACAGUCAGAAAGGUCCUGAGCAUGCCGCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGCAAGGAAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGAAAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAGUCGCAUACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAACUGCUGGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUCAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUCGAACUGGAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCAGGAGAACUGCAGAAGGGAAACGAACUGGCACUGCCGAGCAAGUACGUCAACUUCCUGUACCUGGCAAGCCACUACGAAAAGCUGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUCGUCGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACAGAUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAAACCUGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACAAGCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUGUUCACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUACUUCGACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAGGAAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACAGGACUGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAGACGGAGGAGGAAGCCCGAAGAAGAAGAGA AAGGUCUAG Cas9 mRNAGACAAGAAGUACAGCAUCGGACUGGACAUCGGAACAAACAGCGUCGGAUGG 210 codingGCAGUCAUCACAGACGAAUACAAGGUCCCGAGCAAGAAGUUCAAGGUCCUG sequenceGGAAACACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGAGCACUGCUG usingUUCGACAGCGGAGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCAAGA minimalAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAGGAAAUCUUC uridineAGCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCACAGACUGGAAGAA codons (noAGCUUCCUGGUCGAAGAAGACAAGAAGCACGAAAGACACCCGAUCUUCGGA start orAACAUCGUCGACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUACCAC stop codons;CUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACCUGAGACUGAUC suit able forUACCUGGCACUGGCACACAUGAUCAAGUUCAGAGGACACUUCCUGAUCGAA inclusion inGGAGACCUGAACCCGGACAACAGCGACGUCGACAAGCUGUUCAUCCAGCUG fusionGUCCAGACAUACAACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGA proteinGUCGACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAGCAGAAGACUG codingGAAAACCUGAUCGCACAGCUGCCGGGAGAAAAGAAGAACGGACUGUUCGGA sequence)AACCUGAUCGCACUGAGCCUGGGACUGACACCGAACUUCAAGAGCAACUUCGACCUGGCAGAAGACGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGACGACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCAGACCUGUUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCUGCUGAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGGCACCGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACACCACCAGGACCUGACACUGCUGAAGGCACUGGUCAGACAGCAGCUGCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCAAGAACGGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAGGAAGAAUUCUACAAGUUCAUCAAGCCGAUCCUGGAAAAGAUGGACGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAGACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCCCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAGACAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGAAAAGAUCGAAAAGAUCCUGACAUUCAGAAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAACAGCAGAUUCGCAUGGAUGACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUCGAAGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAAAGAAUGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAAGCACAGCCUGCUGUACGAAUACUUCACAGUCUACAACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAAUGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCAAUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGUCAAGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUCGACAGCGUCGAAAUCAGCGGAGUCGAAGACAGAUUCAACGCAAGCCUGGGAACAUACCACGACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAAGAAAACGAAGACAUCCUGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGAGAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCGACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUCAGAGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCCACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCACAGGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGCAAACCUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGCAGACAGUCAAGGUCGUCGACGAACUGGUCAAGGUCAUGGGAAGACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAAUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGCCAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAGAACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAAGAGACAUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGCGACUACGACGUCGACCACAUCGUCCCGCAGAGCUUCCUGAAGGACGACAGCAUCGACAACAAGGUCCUGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGUCGUCAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCAAAGCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAGGCAGAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAUCAAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAGCACGUCGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUACGACGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACACUGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAGUUCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACACGACGCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAGCUGGAAAGCGAAUUCGUCUACGGAGACUACAAGGUCUACGACGUCAGAAAGAUGAUCGCAAAGAGCGAACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACAGCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAACGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGGAGAAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGACUUCGCAACAGUCAGAAAGGUCCUGAGCAUGCCGCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGCAAGGAAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGAAAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAGUCGCAUACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAACUGCUGGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUCAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUCGAACUGGAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCAGGAGAACUGCAGAAGGGAAACGAACUGGCACUGCCGAGCAAGUACGUCAACUUCCUGUACCUGGCAAGCCACUACGAAAAGCUGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUCGUCGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACAGAUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAAACCUGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACAAGCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUGUUCACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUACUUCGACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAGGAAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACAGGACUGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAGACGGAGGAGGAAGCCCGAAGAAGAAGAGAAAG GUC Cas9 nickaseGACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAAACAGCGUCGGAUGG 211 codingGCAGUCAUCACAGACGAAUACAAGGUCCCGAGCAAGAAGUUCAAGGUCCUG sequenceGGAAACACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGAGCACUGCUG usingUUCGACAGCGGAGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCAAGA minimalAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAGGAAAUCUUC uridineAGCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCACAGACUGGAAGAA codons (noAGCUUCCUGGUCGAAGAAGACAAGAAGCACGAAAGACACCCGAUCUUCGGA start orAACAUCGUCGACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUACCAC stop codons;CUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACCUGAGACUGAUC suitable forUACCUGGCACUGGCACACAUGAUCAAGUUCAGAGGACACUUCCUGAUCGAA inclusion inGGAGACCUGAACCCGGACAACAGCGACGUCGACAAGCUGUUCAUCCAGCUG fusionGUCCAGACAUACAACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGA proteinGUCGACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAGCAGAAGACUG codingGAAAACCUGAUCGCACAGCUGCCGGGAGAAAAGAAGAACGGACUGUUCGGA sequence)AACCUGAUCGCACUGAGCCUGGGACUGACACCGAACUUCAAGAGCAACUUCGACCUGGCAGAAGACGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGACGACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCAGACCUGUUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCUGCUGAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGGCACCGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACACCACCAGGACCUGACACUGCUGAAGGCACUGGUCAGACAGCAGCUGCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCAAGAACGGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAGGAAGAAUUCUACAAGUUCAUCAAGCCGAUCCUGGAAAAGAUGGACGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAGACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCCCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAGACAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGAAAAGAUCGAAAAGAUCCUGACAUUCAGAAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAACAGCAGAUUCGCAUGGAUGACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUCGAAGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAAAGAAUGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAAGCACAGCCUGCUGUACGAAUACUUCACAGUCUACAACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAAUGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCAAUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGUCAAGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUCGACAGCGUCGAAAUCAGCGGAGUCGAAGACAGAUUCAACGCAAGCCUGGGAACAUACCACGACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAAGAAAACGAAGACAUCCUGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGAGAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCGACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUCAGAGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCCACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCACAGGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGCAAACCUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGCAGACAGUCAAGGUCGUCGACGAACUGGUCAAGGUCAUGGGAAGACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAAUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGCCAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAGAACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAAGAGACAUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGCGACUACGACGUCGACCACAUCGUCCCGCAGAGCUUCCUGAAGGACGACAGCAUCGACAACAAGGUCCUGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGUCGUCAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCAAAGCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAGGCAGAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAUCAAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAGCACGUCGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUACGACGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACACUGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAGUUCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACACGACGCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAGCUGGAAAGCGAAUUCGUCUACGGAGACUACAAGGUCUACGACGUCAGAAAGAUGAUCGCAAAGAGCGAACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACAGCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAACGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGGAGAAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGACUUCGCAACAGUCAGAAAGGUCCUGAGCAUGCCGCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGCAAGGAAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGAAAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAGUCGCAUACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAACUGCUGGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUCAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUCGAACUGGAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCAGGAGAACUGCAGAAGGGAAACGAACUGGCACUGCCGAGCAAGUACGUCAACUUCCUGUACCUGGCAAGCCACUACGAAAAGCUGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUCGUCGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACAGAUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAAACCUGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACAAGCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUGUUCACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUACUUCGACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAGGAAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACAGGACUGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAGACGGAGGAGGAAGCCCGAAGAAGAAGAGAAAG GUC dCas9 codingGACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAAACAGCGUCGGAUGG 212 sequenceGCAGUCAUCACAGACGAAUACAAGGUCCCGAGCAAGAAGUUCAAGGUCCUG usingGGAAACACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGAGCACUGCUG minimalUUCGACAGCGGAGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCAAGA uridineAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAGGAAAUCUUC codons (noAGCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCACAGACUGGAAGAA start orAGCUUCCUGGUCGAAGAAGACAAGAAGCACGAAAGACACCCGAUCUUCGGA stop codons;AACAUCGUCGACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUACCAC suitable forCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACCUGAGACUGAUC inclusion inUACCUGGCACUGGCACACAUGAUCAAGUUCAGAGGACACUUCCUGAUCGAA fusionGGAGACCUGAACCCGGACAACAGCGACGUCGACAAGCUGUUCAUCCAGCUG proteinGUCCAGACAUACAACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGA codingGUCGACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAGCAGAAGACUG sequence)GAAAACCUGAUCGCACAGCUGCCGGGAGAAAAGAAGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUGGGACUGACACCGAACUUCAAGAGCAACUUCGACCUGGCAGAAGACGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGACGACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCAGACCUGUUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCUGCUGAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGGCACCGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACACCACCAGGACCUGACACUGCUGAAGGCACUGGUCAGACAGCAGCUGCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCAAGAACGGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAGGAAGAAUUCUACAAGUUCAUCAAGCCGAUCCUGGAAAAGAUGGACGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAGACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCCCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAGACAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGAAAAGAUCGAAAAGAUCCUGACAUUCAGAAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAACAGCAGAUUCGCAUGGAUGACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUCGAAGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAAAGAAUGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAAGCACAGCCUGCUGUACGAAUACUUCACAGUCUACAACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAAUGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCAAUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGUCAAGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUCGACAGCGUCGAAAUCAGCGGAGUCGAAGACAGAUUCAACGCAAGCCUGGGAACAUACCACGACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAAGAAAACGAAGACAUCCUGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGAGAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCGACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUCAGAGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCCACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCACAGGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGCAAACCUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGCAGACAGUCAAGGUCGUCGACGAACUGGUCAAGGUCAUGGGAAGACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAAUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGCCAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAGAACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAAGAGACAUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGCGACUACGACGUCGACGCAAUCGUCCCGCAGAGCUUCCUGAAGGACGACAGCAUCGACAACAAGGUCCUGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGUCGUCAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCAAAGCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAGGCAGAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAUCAAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAGCACGUCGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUACGACGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACACUGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAGUUCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACACGACGCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAGCUGGAAAGCGAAUUCGUCUACGGAGACUACAAGGUCUACGACGUCAGAAAGAUGAUCGCAAAGAGCGAACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACAGCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAACGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGGAGAAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGACUUCGCAACAGUCAGAAAGGUCCUGAGCAUGCCGCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGCAAGGAAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGAAAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAGUCGCAUACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAACUGCUGGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUCAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUCGAACUGGAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCAGGAGAACUGCAGAAGGGAAACGAACUGGCACUGCCGAGCAAGUACGUCAACUUCCUGUACCUGGCAAGCCACUACGAAAAGCUGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUCGUCGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACAGAUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAAACCUGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACAAGCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUGUUCACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUACUUCGACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAGGAAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACAGGACUGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAGACGGAGGAGGAAGCCCGAAGAAGAAGAGAAAG GUC Amino acidMDKKYSIGLDIGINSVGWAVITDEYKVPSKKFKVLGNIDRHSIKKNLIGAL 213 sequence ofLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLE Cas9ESELVEEDKKHERHPIEGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRL (withoutIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS NLS)GVDAKAILSARLSKSRRLENLIAQLPGEKKNGLEGNLIALSLGLIPNEKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLILLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMINFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLEKTNRKVIVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLILTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLIFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLIRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKEDNLIKAERGGLSELDKAGFIKRQLVETRQIIKHVAQILDSRMNIKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGIALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFEKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGESKESILPKRNSDKLIARKKDWDPKKYGGFDSPIVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLINLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD Cas9 mRNAAUGGACAAGAAGUACAGCAUCGGACUGGACAUCGGAACAAACAGCGUCGGA 214 ORF encodingUGGGCAGUCAUCACAGACGAAUACAAGGUCCCGAGCAAGAAGUUCAAGGUC SEQ ID NO:CUGGGAAACACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGAGCACUG 213 usingCUGUUCGACAGCGGAGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCA minimalAGAAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAGGAAAUC uridineUUCAGCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCACAGACUGGAA codons, withGAAAGCUUCCUGGUCGAAGAAGACAAGAAGCACGAAAGACACCCGAUCUUC start andGGAAACAUCGUCGACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUAC stop codonsCACCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACCUGAGACUGAUCUACCUGGCACUGGCACACAUGAUCAAGUUCAGAGGACACUUCCUGAUCGAAGGAGACCUGAACCCGGACAACAGCGACGUCGACAAGCUGUUCAUCCAGCUGGUCCAGACAUACAACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGAGUCGACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAGCAGAAGACUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAAAGAAGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUGGGACUGACACCGAACUUCAAGAGCAACUUCGACCUGGCAGAAGACGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGACGACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCAGACCUGUUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCUGCUGAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGGCACCGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACACCACCAGGACCUGACACUGCUGAAGGCACUGGUCAGACAGCAGCUGCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCAAGAACGGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAGGAAGAAUUCUACAAGUUCAUCAAGCCGAUCCUGGAAAAGAUGGACGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAGACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCCCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAGACAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGAAAAGAUCGAAAAGAUCCUGACAUUCAGAAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAACAGCAGAUUCGCAUGGAUGACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUCGAAGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAAAGAAUGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAAGCACAGCCUGCUGUACGAAUACUUCACAGUCUACAACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAAUGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCAAUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGUCAAGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUCGACAGCGUCGAAAUCAGCGGAGUCGAAGACAGAUUCAACGCAAGCCUGGGAACAUACCACGACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAAGAAAACGAAGACAUCCUGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGAGAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCGACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUCAGAGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCCACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCACAGGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGCAAACCUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGCAGACAGUCAAGGUCGUCGACGAACUGGUCAAGGUCAUGGGAAGACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAAUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGCCAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAGAACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAAGAGACAUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGCGACUACGACGUCGACCACAUCGUCCCGCAGAGCUUCCUGAAGGACGACAGCAUCGACAACAAGGUCCUGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGUCGUCAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCAAAGCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAGGCAGAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAUCAAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAGCACGUCGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUACGACGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACACUGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAGUUCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACACGACGCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAGCUGGAAAGCGAAUUCGUCUACGGAGACUACAAGGUCUACGACGUCAGAAAGAUGAUCGCAAAGAGCGAACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACAGCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAACGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGGAGAAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGACUUCGCAACAGUCAGAAAGGUCCUGAGCAUGCCGCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGCAAGGAAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGAAAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAGUCGCAUACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAACUGCUGGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUCAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUCGAACUGGAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCAGGAGAACUGCAGAAGGGAAACGAACUGGCACUGCCGAGCAAGUACGUCAACUUCCUGUACCUGGCAAGCCACUACGAAAAGCUGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUCGUCGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACAGAUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAAACCUGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACAAGCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUGUUCACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUACUUCGACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAGGAAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACAGGACUGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAGACUAG Cas9 codingGACAAGAAGUACAGCAUCGGACUGGACAUCGGAACAAACAGCGUCGGAUGG 215 sequenceGCAGUCAUCACAGACGAAUACAAGGUCCCGAGCAAGAAGUUCAAGGUCCUG encoding SEQGGAAACACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGAGCACUGCUG ID NO: 213UUCGACAGCGGAGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCAAGA usingAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAGGAAAUCUUC minimalAGCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCACAGACUGGAAGAA uridineAGCUUCCUGGUCGAAGAAGACAAGAAGCACGAAAGACACCCGAUCUUCGGA codons (noAACAUCGUCGACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUACCAC start orCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACCUGAGACUGAUC stop codons;UACCUGGCACUGGCACACAUGAUCAAGUUCAGAGGACACUUCCUGAUCGAA suitable forGGAGACCUGAACCCGGACAACAGCGACGUCGACAAGCUGUUCAUCCAGCUG inclusion inGUCCAGACAUACAACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGA fusionGUCGACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAGCAGAAGACUG proteinGAAAACCUGAUCGCACAGCUGCCGGGAGAAAAGAAGAACGGACUGUUCGGA codingAACCUGAUCGCACUGAGCCUGGGACUGACACCGAACUUCAAGAGCAACUUC sequence)GACCUGGCAGAAGACGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGACGACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCAGACCUGUUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCUGCUGAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGGCACCGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACACCACCAGGACCUGACACUGCUGAAGGCACUGGUCAGACAGCAGCUGCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCAAGAACGGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAGGAAGAAUUCUACAAGUUCAUCAAGCCGAUCCUGGAAAAGAUGGACGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAGACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCCCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAGACAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGAAAAGAUCGAAAAGAUCCUGACAUUCAGAAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAACAGCAGAUUCGCAUGGAUGACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUCGAAGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAAAGAAUGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAAGCACAGCCUGCUGUACGAAUACUUCACAGUCUACAACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAAUGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCAAUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGUCAAGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUCGACAGCGUCGAAAUCAGCGGAGUCGAAGACAGAUUCAACGCAAGCCUGGGAACAUACCACGACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAAGAAAACGAAGACAUCCUGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGAGAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCGACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUCAGAGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCCACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCACAGGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGCAAACCUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGCAGACAGUCAAGGUCGUCGACGAACUGGUCAAGGUCAUGGGAAGACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAAUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGCCAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAGAACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAAGAGACAUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGCGACUACGACGUCGACCACAUCGUCCCGCAGAGCUUCCUGAAGGACGACAGCAUCGACAACAAGGUCCUGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGUCGUCAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCAAAGCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAGGCAGAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAUCAAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAGCACGUCGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUACGACGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACACUGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAGUUCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACACGACGCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAGCUGGAAAGCGAAUUCGUCUACGGAGACUACAAGGUCUACGACGUCAGAAAGAUGAUCGCAAAGAGCGAACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACAGCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAACGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGGAGAAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGACUUCGCAACAGUCAGAAAGGUCCUGAGCAUGCCGCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGCAAGGAAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGAAAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAGUCGCAUACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAACUGCUGGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUCAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUCGAACUGGAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCAGGAGAACUGCAGAAGGGAAACGAACUGGCACUGCCGAGCAAGUACGUCAACUUCCUGUACCUGGCAAGCCACUACGAAAAGCUGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUCGUCGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACAGAUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAAACCUGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACAAGCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUGUUCACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUACUUCGACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAGGAAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACAGGACUGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAGAC Amino acidMDKKYSIGLAIGINSVGWAVITDEYKVPSKKFKVLGNIDRHSIKKNLIGAL 216 sequence ofLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLE Cas9 nickaseESELVEEDKKHERHPIEGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRL (withoutIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS NLS)GVDAKAILSARLSKSRRLENLIAQLPGEKKNGLEGNLIALSLGLIPNEKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLILLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMINFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLEKTNRKVIVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLILTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLIFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLIRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKEDNLIKAERGGLSELDKAGFIKRQLVETRQIIKHVAQILDSRMNIKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGIALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFEKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGESKESILPKRNSDKLIARKKDWDPKKYGGFDSPIVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLINLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD Cas9 nickaseAUGGACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAAACAGCGUCGGA 217 mRNA ORFUGGGCAGUCAUCACAGACGAAUACAAGGUCCCGAGCAAGAAGUUCAAGGUC encoding SEQCUGGGAAACACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGAGCACUG ID NO: 216CUGUUCGACAGCGGAGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCA usingAGAAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAGGAAAUC minimalUUCAGCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCACAGACUGGAA uridineGAAAGCUUCCUGGUCGAAGAAGACAAGAAGCACGAAAGACACCCGAUCUUC codons asGGAAACAUCGUCGACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUAC listed inCACCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACCUGAGACUG Table 3,AUCUACCUGGCACUGGCACACAUGAUCAAGUUCAGAGGACACUUCCUGAUC with startGAAGGAGACCUGAACCCGGACAACAGCGACGUCGACAAGCUGUUCAUCCAG and stopCUGGUCCAGACAUACAACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGC codonsGGAGUCGACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAGCAGAAGACUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAAAGAAGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUGGGACUGACACCGAACUUCAAGAGCAACUUCGACCUGGCAGAAGACGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGACGACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCAGACCUGUUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCUGCUGAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGGCACCGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACACCACCAGGACCUGACACUGCUGAAGGCACUGGUCAGACAGCAGCUGCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCAAGAACGGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAGGAAGAAUUCUACAAGUUCAUCAAGCCGAUCCUGGAAAAGAUGGACGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAGACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCCCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAGACAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGAAAAGAUCGAAAAGAUCCUGACAUUCAGAAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAACAGCAGAUUCGCAUGGAUGACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUCGAAGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAAAGAAUGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAAGCACAGCCUGCUGUACGAAUACUUCACAGUCUACAACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAAUGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCAAUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGUCAAGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUCGACAGCGUCGAAAUCAGCGGAGUCGAAGACAGAUUCAACGCAAGCCUGGGAACAUACCACGACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAAGAAAACGAAGACAUCCUGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGAGAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCGACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUCAGAGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCCACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCACAGGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGCAAACCUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGCAGACAGUCAAGGUCGUCGACGAACUGGUCAAGGUCAUGGGAAGACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAAUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGCCAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAGAACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAAGAGACAUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGCGACUACGACGUCGACCACAUCGUCCCGCAGAGCUUCCUGAAGGACGACAGCAUCGACAACAAGGUCCUGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGUCGUCAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCAAAGCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAGGCAGAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAUCAAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAGCACGUCGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUACGACGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACACUGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAGUUCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACACGACGCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAGCUGGAAAGCGAAUUCGUCUACGGAGACUACAAGGUCUACGACGUCAGAAAGAUGAUCGCAAAGAGCGAACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACAGCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAACGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGGAGAAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGACUUCGCAACAGUCAGAAAGGUCCUGAGCAUGCCGCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGCAAGGAAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGAAAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAGUCGCAUACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAACUGCUGGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUCAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUCGAACUGGAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCAGGAGAACUGCAGAAGGGAAACGAACUGGCACUGCCGAGCAAGUACGUCAACUUCCUGUACCUGGCAAGCCACUACGAAAAGCUGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUCGUCGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACAGAUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAAACCUGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACAAGCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUGUUCACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUACUUCGACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAGGAAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACAGGACUGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAGACUAG Cas9 nickaseGACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAAACAGCGUCGGAUGG 218 codingGCAGUCAUCACAGACGAAUACAAGGUCCCGAGCAAGAAGUUCAAGGUCCUG sequenceGGAAACACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGAGCACUGCUG encoding SEQUUCGACAGCGGAGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCAAGA ID NO: 216AGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAGGAAAUCUUC usingAGCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCACAGACUGGAAGAA minimalAGCUUCCUGGUCGAAGAAGACAAGAAGCACGAAAGACACCCGAUCUUCGGA uridineAACAUCGUCGACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUACCAC codons asCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACCUGAGACUGAUC listed inUACCUGGCACUGGCACACAUGAUCAAGUUCAGAGGACACUUCCUGAUCGAA Table 3 (noGGAGACCUGAACCCGGACAACAGCGACGUCGACAAGCUGUUCAUCCAGCUG start orGUCCAGACAUACAACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGA stop codons;GUCGACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAGCAGAAGACUG suitable forGAAAACCUGAUCGCACAGCUGCCGGGAGAAAAGAAGAACGGACUGUUCGGA inclusion inAACCUGAUCGCACUGAGCCUGGGACUGACACCGAACUUCAAGAGCAACUUC fusionGACCUGGCAGAAGACGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGAC proteinGACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCAGACCUGUUC codingCUGGCAGCAAAGAACCUGAGCGACGCAAUCCUGCUGAGCGACAUCCUGAGA sequence)GUCAACACAGAAAUCACAAAGGCACCGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACACCACCAGGACCUGACACUGCUGAAGGCACUGGUCAGACAGCAGCUGCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCAAGAACGGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAGGAAGAAUUCUACAAGUUCAUCAAGCCGAUCCUGGAAAAGAUGGACGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAGACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCCCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAGACAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGAAAAGAUCGAAAAGAUCCUGACAUUCAGAAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAACAGCAGAUUCGCAUGGAUGACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUCGAAGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAAAGAAUGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAAGCACAGCCUGCUGUACGAAUACUUCACAGUCUACAACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAAUGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCAAUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGUCAAGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUCGACAGCGUCGAAAUCAGCGGAGUCGAAGACAGAUUCAACGCAAGCCUGGGAACAUACCACGACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAAGAAAACGAAGACAUCCUGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGAGAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCGACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUCAGAGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCCACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCACAGGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGCAAACCUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGCAGACAGUCAAGGUCGUCGACGAACUGGUCAAGGUCAUGGGAAGACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAAUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGCCAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAGAACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAAGAGACAUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGCGACUACGACGUCGACCACAUCGUCCCGCAGAGCUUCCUGAAGGACGACAGCAUCGACAACAAGGUCCUGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGUCGUCAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCAAAGCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAGGCAGAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAUCAAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAGCACGUCGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUACGACGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACACUGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAGUUCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACACGACGCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAGCUGGAAAGCGAAUUCGUCUACGGAGACUACAAGGUCUACGACGUCAGAAAGAUGAUCGCAAAGAGCGAACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACAGCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAACGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGGAGAAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGACUUCGCAACAGUCAGAAAGGUCCUGAGCAUGCCGCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGCAAGGAAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGAAAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAGUCGCAUACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAACUGCUGGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUCAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUCGAACUGGAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCAGGAGAACUGCAGAAGGGAAACGAACUGGCACUGCCGAGCAAGUACGUCAACUUCCUGUACCUGGCAAGCCACUACGAAAAGCUGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUCGUCGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACAGAUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAAACCUGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACAAGCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUGUUCACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUACUUCGACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAGGAAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACAGGACUGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAGAC Amino acidMDKKYSIGLAIGINSVGWAVITDEYKVPSKKFKVLGNIDRHSIKKNLIGAL 219 sequence ofLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLE dCas9ESELVEEDKKHERHPIEGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRL (withoutIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS NLS)GVDAKAILSARLSKSRRLENLIAQLPGEKKNGLEGNLIALSLGLIPNEKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLILLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMINFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLEKTNRKVIVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLILTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLIFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLIRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKEDNLIKAERGGLSELDKAGFIKRQLVETRQIIKHVAQILDSRMNIKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGIALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFEKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGESKESILPKRNSDKLIARKKDWDPKKYGGFDSPIVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD dCas9 mRNAAUGGACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAAACAGCGUCGGA 220 ORF encodingUGGGCAGUCAUCACAGACGAAUACAAGGUCCCGAGCAAGAAGUUCAAGGUC SEQ ID NO:CUGGGAAACACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGAGCACUG 219 usingCUGUUCGACAGCGGAGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCA minimalAGAAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAGGAAAUC uridineUUCAGCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCACAGACUGGAA codons asGAAAGCUUCCUGGUCGAAGAAGACAAGAAGCACGAAAGACACCCGAUCUUC listed inGGAAACAUCGUCGACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUAC Table 3,CACCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACCUGAGACUG with startAUCUACCUGGCACUGGCACACAUGAUCAAGUUCAGAGGACACUUCCUGAUC and stopGAAGGAGACCUGAACCCGGACAACAGCGACGUCGACAAGCUGUUCAUCCAG codonsCUGGUCCAGACAUACAACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGAGUCGACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAGCAGAAGACUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAAAGAAGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUGGGACUGACACCGAACUUCAAGAGCAACUUCGACCUGGCAGAAGACGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGACGACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCAGACCUGUUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCUGCUGAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGGCACCGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACACCACCAGGACCUGACACUGCUGAAGGCACUGGUCAGACAGCAGCUGCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCAAGAACGGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAGGAAGAAUUCUACAAGUUCAUCAAGCCGAUCCUGGAAAAGAUGGACGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAGACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCCCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAGACAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGAAAAGAUCGAAAAGAUCCUGACAUUCAGAAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAACAGCAGAUUCGCAUGGAUGACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUCGAAGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAAAGAAUGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAAGCACAGCCUGCUGUACGAAUACUUCACAGUCUACAACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAAUGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCAAUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGUCAAGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUCGACAGCGUCGAAAUCAGCGGAGUCGAAGACAGAUUCAACGCAAGCCUGGGAACAUACCACGACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAAGAAAACGAAGACAUCCUGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGAGAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCGACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUCAGAGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCCACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCACAGGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGCAAACCUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGCAGACAGUCAAGGUCGUCGACGAACUGGUCAAGGUCAUGGGAAGACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAAUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGCCAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAGAACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAAGAGACAUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGCGACUACGACGUCGACGCAAUCGUCCCGCAGAGCUUCCUGAAGGACGACAGCAUCGACAACAAGGUCCUGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGUCGUCAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCAAAGCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAGGCAGAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAUCAAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAGCACGUCGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUACGACGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACACUGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAGUUCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACACGACGCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAGCUGGAAAGCGAAUUCGUCUACGGAGACUACAAGGUCUACGACGUCAGAAAGAUGAUCGCAAAGAGCGAACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACAGCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAACGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGGAGAAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGACUUCGCAACAGUCAGAAAGGUCCUGAGCAUGCCGCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGCAAGGAAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGAAAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAGUCGCAUACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAACUGCUGGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUCAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUCGAACUGGAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCAGGAGAACUGCAGAAGGGAAACGAACUGGCACUGCCGAGCAAGUACGUCAACUUCCUGUACCUGGCAAGCCACUACGAAAAGCUGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUCGUCGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACAGAUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAAACCUGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACAAGCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUGUUCACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUACUUCGACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAGGAAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACAGGACUGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAGACUAG dCas9 codingGACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAAACAGCGUCGGAUGG 221 sequenceGCAGUCAUCACAGACGAAUACAAGGUCCCGAGCAAGAAGUUCAAGGUCCUG encoding SEQGGAAACACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGAGCACUGCUG ID NO: 219UUCGACAGCGGAGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCAAGA usingAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAGGAAAUCUUC minimalAGCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCACAGACUGGAAGAA uridineAGCUUCCUGGUCGAAGAAGACAAGAAGCACGAAAGACACCCGAUCUUCGGA codons asAACAUCGUCGACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUACCAC listed inCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACCUGAGACUGAUC Table 3 (noUACCUGGCACUGGCACACAUGAUCAAGUUCAGAGGACACUUCCUGAUCGAA start orGGAGACCUGAACCCGGACAACAGCGACGUCGACAAGCUGUUCAUCCAGCUG stop codons;GUCCAGACAUACAACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGA suitable forGUCGACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAGCAGAAGACUG inclusion inGAAAACCUGAUCGCACAGCUGCCGGGAGAAAAGAAGAACGGACUGUUCGGA fusionAACCUGAUCGCACUGAGCCUGGGACUGACACCGAACUUCAAGAGCAACUUC proteinGACCUGGCAGAAGACGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGAC codingGACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCAGACCUGUUC sequence)CUGGCAGCAAAGAACCUGAGCGACGCAAUCCUGCUGAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGGCACCGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACACCACCAGGACCUGACACUGCUGAAGGCACUGGUCAGACAGCAGCUGCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCAAGAACGGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAGGAAGAAUUCUACAAGUUCAUCAAGCCGAUCCUGGAAAAGAUGGACGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAGACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCCCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAGACAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGAAAAGAUCGAAAAGAUCCUGACAUUCAGAAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAACAGCAGAUUCGCAUGGAUGACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUCGAAGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAAAGAAUGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAAGCACAGCCUGCUGUACGAAUACUUCACAGUCUACAACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAAUGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCAAUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGUCAAGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUCGACAGCGUCGAAAUCAGCGGAGUCGAAGACAGAUUCAACGCAAGCCUGGGAACAUACCACGACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAAGAAAACGAAGACAUCCUGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGAGAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCGACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUCAGAGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCCACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCACAGGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGCAAACCUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGCAGACAGUCAAGGUCGUCGACGAACUGGUCAAGGUCAUGGGAAGACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAAUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGCCAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAGAACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAAGAGACAUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGCGACUACGACGUCGACGCAAUCGUCCCGCAGAGCUUCCUGAAGGACGACAGCAUCGACAACAAGGUCCUGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGUCGUCAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCAAAGCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAGGCAGAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAUCAAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAGCACGUCGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUACGACGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACACUGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAGUUCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACACGACGCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAGCUGGAAAGCGAAUUCGUCUACGGAGACUACAAGGUCUACGACGUCAGAAAGAUGAUCGCAAAGAGCGAACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACAGCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAACGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGGAGAAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGACUUCGCAACAGUCAGAAAGGUCCUGAGCAUGCCGCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGCAAGGAAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGAAAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAGUCGCAUACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAACUGCUGGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUCAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUCGAACUGGAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCAGGAGAACUGCAGAAGGGAAACGAACUGGCACUGCCGAGCAAGUACGUCAACUUCCUGUACCUGGCAAGCCACUACGAAAAGCUGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUCGUCGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACAGAUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAAACCUGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACAAGCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUGUUCACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUACUUCGACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAGGAAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACAGGACUGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAGACGGAGGAGGAAGC Amino acidMDKKYSIGLDIGINSVGWAVITDEYKVPSKKFKVLGNIDRHSIKKNLIGAL 222 sequence ofLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLE Cas9 withESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRL two nuclearIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS localizationGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLIPNFKSN signalsFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDIL (2xNLS) asRVNTEITKAPLSASMIKRYDEHHQDLILLKALVRQQLPEKYKEIFFDQSKN the C-GYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNG terminalSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN amino acidsSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMINFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKINRKVIVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLILTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLIFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLIRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLIKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFEKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDGSGSPKKKR KVDGSPKKKRKVDSGCas9 mRNA AUGGACAAGAAGUACAGCAUCGGACUGGACAUCGGAACAAACAGCGUCGGA 223ORF encoding UGGGCAGUCAUCACAGACGAAUACAAGGUCCCGAGCAAGAAGUUCAAGGUCSEQ ID NO: CUGGGAAACACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGAGCACUG 222 usingCUGUUCGACAGCGGAGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCA minimalAGAAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAGGAAAUC uridineUUCAGCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCACAGACUGGAA codons, withGAAAGCUUCCUGGUCGAAGAAGACAAGAAGCACGAAAGACACCCGAUCUUC start andGGAAACAUCGUCGACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUAC stop codonsCACCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACCUGAGACUGAUCUACCUGGCACUGGCACACAUGAUCAAGUUCAGAGGACACUUCCUGAUCGAAGGAGACCUGAACCCGGACAACAGCGACGUCGACAAGCUGUUCAUCCAGCUGGUCCAGACAUACAACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGAGUCGACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAGCAGAAGACUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAAAGAAGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUGGGACUGACACCGAACUUCAAGAGCAACUUCGACCUGGCAGAAGACGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGACGACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCAGACCUGUUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCUGCUGAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGGCACCGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACACCACCAGGACCUGACACUGCUGAAGGCACUGGUCAGACAGCAGCUGCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCAAGAACGGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAGGAAGAAUUCUACAAGUUCAUCAAGCCGAUCCUGGAAAAGAUGGACGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAGACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCCCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAGACAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGAAAAGAUCGAAAAGAUCCUGACAUUCAGAAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAACAGCAGAUUCGCAUGGAUGACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUCGAAGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAAAGAAUGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAAGCACAGCCUGCUGUACGAAUACUUCACAGUCUACAACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAAUGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCAAUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGUCAAGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUCGACAGCGUCGAAAUCAGCGGAGUCGAAGACAGAUUCAACGCAAGCCUGGGAACAUACCACGACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAAGAAAACGAAGACAUCCUGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGAGAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCGACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUCAGAGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCCACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCACAGGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGCAAACCUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGCAGACAGUCAAGGUCGUCGACGAACUGGUCAAGGUCAUGGGAAGACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAAUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGCCAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAGAACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAAGAGACAUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGCGACUACGACGUCGACCACAUCGUCCCGCAGAGCUUCCUGAAGGACGACAGCAUCGACAACAAGGUCCUGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGUCGUCAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCAAAGCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAGGCAGAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAUCAAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAGCACGUCGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUACGACGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACACUGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAGUUCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACACGACGCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAGCUGGAAAGCGAAUUCGUCUACGGAGACUACAAGGUCUACGACGUCAGAAAGAUGAUCGCAAAGAGCGAACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACAGCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAACGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGGAGAAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGACUUCGCAACAGUCAGAAAGGUCCUGAGCAUGCCGCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGCAAGGAAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGAAAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAGUCGCAUACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAACUGCUGGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUCAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUCGAACUGGAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCAGGAGAACUGCAGAAGGGAAACGAACUGGCACUGCCGAGCAAGUACGUCAACUUCCUGUACCUGGCAAGCCACUACGAAAAGCUGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUCGUCGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACAGAUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAAACCUGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACAAGCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUGUUCACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUACUUCGACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAGGAAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACAGGACUGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAGACGGAGGAGGAAGCCCGAAGAAGAAGAGAAAGGUCCCGAAGAAGAAGAGAAAGGUCGGAAGCGGAAGCCCGAAGAAGAAGAGAAAGGUCGACGGAAGCCCGAAGAAGAAGAGAAAGGUCGACAGCGGAUAG Cas9 codingGACAAGAAGUACAGCAUCGGACUGGACAUCGGAACAAACAGCGUCGGAUGG 224 sequence SEQGCAGUCAUCACAGACGAAUACAAGGUCCCGAGCAAGAAGUUCAAGGUCCUG encodingGGAAACACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGAGCACUGCUG ID NO: 222UUCGACAGCGGAGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCAAGA usingAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAGGAAAUCUUC minimalAGCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCACAGACUGGAAGAA uridineAGCUUCCUGGUCGAAGAAGACAAGAAGCACGAAAGACACCCGAUCUUCGGA codons (noAACAUCGUCGACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUACCAC start orCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACCUGAGACUGAUC stop codons;UACCUGGCACUGGCACACAUGAUCAAGUUCAGAGGACACUUCCUGAUCGAA suitable forGGAGACCUGAACCCGGACAACAGCGACGUCGACAAGCUGUUCAUCCAGCUG inclusion inGUCCAGACAUACAACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGA fusionGUCGACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAGCAGAAGACUG proteinGAAAACCUGAUCGCACAGCUGCCGGGAGAAAAGAAGAACGGACUGUUCGGA codingAACCUGAUCGCACUGAGCCUGGGACUGACACCGAACUUCAAGAGCAACUUC sequence)GACCUGGCAGAAGACGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGACGACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCAGACCUGUUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCUGCUGAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGGCACCGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACACCACCAGGACCUGACACUGCUGAAGGCACUGGUCAGACAGCAGCUGCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCAAGAACGGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAGGAAGAAUUCUACAAGUUCAUCAAGCCGAUCCUGGAAAAGAUGGACGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAGACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCCCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAGACAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGAAAAGAUCGAAAAGAUCCUGACAUUCAGAAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAACAGCAGAUUCGCAUGGAUGACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUCGAAGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAAAGAAUGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAAGCACAGCCUGCUGUACGAAUACUUCACAGUCUACAACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAAUGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCAAUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGUCAAGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUCGACAGCGUCGAAAUCAGCGGAGUCGAAGACAGAUUCAACGCAAGCCUGGGAACAUACCACGACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAAGAAAACGAAGACAUCCUGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGAGAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCGACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUCAGAGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCCACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCACAGGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGCAAACCUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGCAGACAGUCAAGGUCGUCGACGAACUGGUCAAGGUCAUGGGAAGACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAAUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGCCAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAGAACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAAGAGACAUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGCGACUACGACGUCGACCACAUCGUCCCGCAGAGCUUCCUGAAGGACGACAGCAUCGACAACAAGGUCCUGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGUCGUCAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCAAAGCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAGGCAGAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAUCAAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAGCACGUCGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUACGACGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACACUGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAGUUCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACACGACGCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAGCUGGAAAGCGAAUUCGUCUACGGAGACUACAAGGUCUACGACGUCAGAAAGAUGAUCGCAAAGAGCGAACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACAGCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAACGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGGAGAAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGACUUCGCAACAGUCAGAAAGGUCCUGAGCAUGCCGCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGCAAGGAAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGAAAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAGUCGCAUACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAACUGCUGGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUCAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUCGAACUGGAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCAGGAGAACUGCAGAAGGGAAACGAACUGGCACUGCCGAGCAAGUACGUCAACUUCCUGUACCUGGCAAGCCACUACGAAAAGCUGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUCGUCGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACAGAUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAAACCUGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACAAGCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUGUUCACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUACUUCGACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAGGAAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACAGGACUGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAGACGGAGGAGGAAGCCCGAAGAAGAAGAGAAAGGUCCCGAAGAAGAAGAGAAAGGUCGGAAGCGGAAGCCCGAAGAAGAAGAGAAAGGUCGACGGAAGCCCGAAGAAGAAGAGAAAGGUCGACAGCGGA Amino acidMDKKYSIGLAIGINSVGWAVITDEYKVPSKKFKVLGNIDRHSIKKNLIGAL 225 sequence ofLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLE Cas9 nickaseESELVEEDKKHERHPIEGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRL with twoIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS nuclearGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLEGNLIALSLGLIPNEKSN localizationFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDIL signals asRVNTEITKAPLSASMIKRYDEHHQDLILLKALVRQQLPEKYKEIFFDQSKN the C-GYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNG terminalSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN amino acidsSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMINFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLEKTNRKVIVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLILTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLIFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLIRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKEDNLIKAERGGLSELDKAGFIKRQLVETRQIIKHVAQILDSRMNIKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGIALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFEKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGESKESILPKRNSDKLIARKKDWDPKKYGGFDSPIVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLINLGAPAAFKYFDTTIDRKRYISTKEVLDATLIHQSITGLYETRIDLSQLGGDGSGSPKKKR KVDGSPKKKRKVDSGCas9 nickase AUGGACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAAACAGCGUCGGA 226mRNA ORF UGGGCAGUCAUCACAGACGAAUACAAGGUCCCGAGCAAGAAGUUCAAGGUCencoding SEQ CUGGGAAACACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGAGCACUGID NO: 25 CUGUUCGACAGCGGAGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCA usingAGAAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAGGAAAUC minimalUUCAGCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCACAGACUGGAA uridineGAAAGCUUCCUGGUCGAAGAAGACAAGAAGCACGAAAGACACCCGAUCUUC codons asGGAAACAUCGUCGACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUAC listed inCACCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACCUGAGACUG Table 3,AUCUACCUGGCACUGGCACACAUGAUCAAGUUCAGAGGACACUUCCUGAUC with startGAAGGAGACCUGAACCCGGACAACAGCGACGUCGACAAGCUGUUCAUCCAG and stopCUGGUCCAGACAUACAACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGC codonsGGAGUCGACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAGCAGAAGACUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAAAGAAGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUGGGACUGACACCGAACUUCAAGAGCAACUUCGACCUGGCAGAAGACGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGACGACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCAGACCUGUUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCUGCUGAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGGCACCGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACACCACCAGGACCUGACACUGCUGAAGGCACUGGUCAGACAGCAGCUGCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCAAGAACGGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAGGAAGAAUUCUACAAGUUCAUCAAGCCGAUCCUGGAAAAGAUGGACGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAGACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCCCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAGACAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGAAAAGAUCGAAAAGAUCCUGACAUUCAGAAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAACAGCAGAUUCGCAUGGAUGACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUCGAAGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAAAGAAUGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAAGCACAGCCUGCUGUACGAAUACUUCACAGUCUACAACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAAUGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCAAUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGUCAAGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUCGACAGCGUCGAAAUCAGCGGAGUCGAAGACAGAUUCAACGCAAGCCUGGGAACAUACCACGACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAAGAAAACGAAGACAUCCUGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGAGAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCGACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUCAGAGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCCACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCACAGGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGCAAACCUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGCAGACAGUCAAGGUCGUCGACGAACUGGUCAAGGUCAUGGGAAGACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAAUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGCCAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAGAACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAAGAGACAUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGCGACUACGACGUCGACCACAUCGUCCCGCAGAGCUUCCUGAAGGACGACAGCAUCGACAACAAGGUCCUGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGUCGUCAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCAAAGCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAGGCAGAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAUCAAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAGCACGUCGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUACGACGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACACUGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAGUUCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACACGACGCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAGCUGGAAAGCGAAUUCGUCUACGGAGACUACAAGGUCUACGACGUCAGAAAGAUGAUCGCAAAGAGCGAACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACAGCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAACGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGGAGAAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGACUUCGCAACAGUCAGAAAGGUCCUGAGCAUGCCGCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGCAAGGAAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGAAAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAGUCGCAUACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAACUGCUGGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUCAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUCGAACUGGAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCAGGAGAACUGCAGAAGGGAAACGAACUGGCACUGCCGAGCAAGUACGUCAACUUCCUGUACCUGGCAAGCCACUACGAAAAGCUGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUCGUCGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACAGAUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAAACCUGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACAAGCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUGUUCACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUACUUCGACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAGGAAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACAGGACUGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAGACGGAAGCGGAAGCCCGAAGAAGAAGAGAAAGGUCGACGGAAGCCCGAAGAAGAAGAGAAAGGUCGACAGCGGAUAG Cas9 nickaseGACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAAACAGCGUCGGAUGG 227 codingGCAGUCAUCACAGACGAAUACAAGGUCCCGAGCAAGAAGUUCAAGGUCCUG sequenceGGAAACACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGAGCACUGCUG encoding SEQUUCGACAGGGAGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCAAGA ID NO: 25AGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAGGAAAUCUUC usingAGCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCACAGACUGGAAGAA minimalAGCUUCCUGGUCGAAGAAGACAAGAAGCACGAAAGACACCCGAUCUUCGGA uridineAACAUCGUCGACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUACCAC codons (noCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACCUGAGACUGAUC start orUACCUGGCACUGGCACACAUGAUCAAGUUCAGAGGACACUUCCUGAUCGAA stop codons;GGAGACCUGAACCCGGACAACAGCGACGUCGACAAGCUGUUCAUCCAGCUG suitable forGUCCAGACAUACAACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGA inclusion inGUCGACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAGCAGAAGACUG fusionGAAAACCUGAUCGCACAGCUGCCGGGAGAAAAGAAGAACGGACUGUUCGGA proteinAACCUGAUCGCACUGAGCCUGGGACUGACACCGAACUUCAAGAGCAACUUC codingGACCUGGCAGAAGACGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGAC sequence)GACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCAGACCUGUUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCUGCUGAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGGCACCGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACACCACCAGGACCUGACACUGCUGAAGGCACUGGUCAGACAGCAGCUGCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCAAGAACGGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAGGAAGAAUUCUACAAGUUCAUCAAGCCGAUCCUGGAAAAGAUGGACGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAGACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCCCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAGACAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGAAAAGAUCGAAAAGAUCCUGACAUUCAGAAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAACAGCAGAUUCGCAUGGAUGACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUCGAAGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAAAGAAUGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAAGCACAGCCUGCUGUACGAAUACUUCACAGUCUACAACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAAUGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCAAUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGUCAAGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUCGACAGCGUCGAAAUCAGCGGAGUCGAAGACAGAUUCAACGCAAGCCUGGGAACAUACCACGACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAAGAAAACGAAGACAUCCUGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGAGAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCGACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUCAGAGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCCACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCACAGGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGCAAACCUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGCAGACAGUCAAGGUCGUCGACGAACUGGUCAAGGUCAUGGGAAGACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAAUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGCCAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAGAACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAAGAGACAUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGCGACUACGACGUCGACCACAUCGUCCCGCAGAGCUUCCUGAAGGACGACAGCAUCGACAACAAGGUCCUGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGUCGUCAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCAAAGCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAGGCAGAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAUCAAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAGCACGUCGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUACGACGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACACUGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAGUUCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACACGACGCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAGCUGGAAAGCGAAUUCGUCUACGGAGACUACAAGGUCUACGACGUCAGAAAGAUGAUCGCAAAGAGCGAACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACAGCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAACGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGGAGAAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGACUUCGCAACAGUCAGAAAGGUCCUGAGCAUGCCGCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGCAAGGAAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGAAAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAGUCGCAUACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAACUGCUGGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUCAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUCGAACUGGAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCAGGAGAACUGCAGAAGGGAAACGAACUGGCACUGCCGAGCAAGUACGUCAACUUCCUGUACCUGGCAAGCCACUACGAAAAGCUGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUCGUCGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACAGAUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAAACCUGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACAAGCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUGUUCACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUACUUCGACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAGGAAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACAGGACUGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAGACGGAAGCGGAAGCCCGAAGAAGAAGAGAAAGGUCGACGGAAGCCCGAAGAAGAAGAGAAAGGUCGACAGCGGA Amino acidMDKKYSIGLAIGINSVGWAVITDEYKVPSKKFKVLGNIDRHSIKKNLIGAL 228 sequence ofLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLE dCas9 withESELVEEDKKHERHPIEGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRL two nuclearIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS localizationGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLEGNLIALSLGLIPNEKSN signals asFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDIL the C-RVNTEITKAPLSASMIKRYDEHHQDLILLKALVRQQLPEKYKEIFFDQSKN terminalGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNG amino acidsSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMINFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLEKTNRKVIVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLILTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLIFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLIRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKEDNLIKAERGGLSELDKAGFIKRQLVETRQIIKHVAQILDSRMNIKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGIALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFEKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGESKESILPKRNSDKLIARKKDWDPKKYGGFDSPIVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLINLGAPAAFKYFDTTIDRKRYISTKEVLDATLIHQSITGLYETRIDLSQLGGDGSGSPKKKR KVDGSPKKKRKVDSGdCas9 mRNA AUGGACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAAACAGCGUCGGA 229ORF encoding UGGGCAGUCAUCACAGACGAAUACAAGGUCCCGAGCAAGAAGUUCAAGGUCSEQ ID NO: CUGGGAAACACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGAGCACUG 228 usingCUGUUCGACAGCGGAGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCA minimalAGAAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAGGAAAUC uridineUUCAGCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCACAGACUGGAA codons, withGAAAGCUUCCUGGUCGAAGAAGACAAGAAGCACGAAAGACACCCGAUCUUC start andGGAAACAUCGUCGACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUAC stop codonsCACCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACCUGAGACUGAUCUACCUGGCACUGGCACACAUGAUCAAGUUCAGAGGACACUUCCUGAUCGAAGGAGACCUGAACCCGGACAACAGCGACGUCGACAAGCUGUUCAUCCAGCUGGUCCAGACAUACAACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGAGUCGACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAGCAGAAGACUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAAAGAAGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUGGGACUGACACCGAACUUCAAGAGCAACUUCGACCUGGCAGAAGACGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGACGACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCAGACCUGUUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCUGCUGAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGGCACCGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACACCACCAGGACCUGACACUGCUGAAGGCACUGGUCAGACAGCAGCUGCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCAAGAACGGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAGGAAGAAUUCUACAAGUUCAUCAAGCCGAUCCUGGAAAAGAUGGACGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAGACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCCCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAGACAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGAAAAGAUCGAAAAGAUCCUGACAUUCAGAAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAACAGCAGAUUCGCAUGGAUGACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUCGAAGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAAAGAAUGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAAGCACAGCCUGCUGUACGAAUACUUCACAGUCUACAACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAAUGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCAAUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGUCAAGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUCGACAGCGUCGAAAUCAGCGGAGUCGAAGACAGAUUCAACGCAAGCCUGGGAACAUACCACGACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAAGAAAACGAAGACAUCCUGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGAGAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCGACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUCAGAGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCCACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCACAGGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGCAAACCUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGCAGACAGUCAAGGUCGUCGACGAACUGGUCAAGGUCAUGGGAAGACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAAUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGCCAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAGAACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAAGAGACAUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGCGACUACGACGUCGACGCAAUCGUCCCGCAGAGCUUCCUGAAGGACGACAGCAUCGACAACAAGGUCCUGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGUCGUCAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCAAAGCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAGGCAGAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAUCAAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAGCACGUCGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUACGACGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACACUGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAGUUCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACACGACGCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAGCUGGAAAGCGAAUUCGUCUACGGAGACUACAAGGUCUACGACGUCAGAAAGAUGAUCGCAAAGAGCGAACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACAGCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAACGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGGAGAAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGACUUCGCAACAGUCAGAAAGGUCCUGAGCAUGCCGCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGCAAGGAAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGAAAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAGUCGCAUACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAACUGCUGGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUCAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUCGAACUGGAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCAGGAGAACUGCAGAAGGGAAACGAACUGGCACUGCCGAGCAAGUACGUCAACUUCCUGUACCUGGCAAGCCACUACGAAAAGCUGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUCGUCGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACAGAUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAAACCUGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACAAGCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUGUUCACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUACUUCGACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAGGAAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACAGGACUGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAGACGGAAGCGGAAGCCCGAAGAAGAAGAGAAAGGUCGACGGAAGCCCGAAGAAGAAGAGAAAGGUCGACAGCGGAUAG dCas9 codingGACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAAACAGCGUCGGAUGG 230 sequenceGCAGUCAUCACAGACGAAUACAAGGUCCCGAGCAAGAAGUUCAAGGUCCUG encoding SEQGGAAACACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGAGCACUGCUG ID NO: 228UUCGACAGCGGAGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCAAGA usingAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAGGAAAUCUUC minimalAGCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCACAGACUGGAAGAA uridineAGCUUCCUGGUCGAAGAAGACAAGAAGCACGAAAGACACCCGAUCUUCGGA codons (noAACAUCGUCGACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUACCAC start orCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACCUGAGACUGAUC stop codons;UACCUGGCACUGGCACACAUGAUCAAGUUCAGAGGACACUUCCUGAUCGAA suitable forGGAGACCUGAACCCGGACAACAGCGACGUCGACAAGCUGUUCAUCCAGCUG inclusion inGUCCAGACAUACAACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGA fusionGUCGACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAGCAGAAGACUG proteinGAAAACCUGAUCGCACAGCUGCCGGGAGAAAAGAAGAACGGACUGUUCGGA codingAACCUGAUCGCACUGAGCCUGGGACUGACACCGAACUUCAAGAGCAACUUC sequence)GACCUGGCAGAAGACGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGACGACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCAGACCUGUUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCUGCUGAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGGCACCGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACACCACCAGGACCUGACACUGCUGAAGGCACUGGUCAGACAGCAGCUGCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCAAGAACGGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAGGAAGAAUUCUACAAGUUCAUCAAGCCGAUCCUGGAAAAGAUGGACGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAGACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCCCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAGACAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGAAAAGAUCGAAAAGAUCCUGACAUUCAGAAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAACAGCAGAUUCGCAUGGAUGACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUCGAAGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAAAGAAUGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAAGCACAGCCUGCUGUACGAAUACUUCACAGUCUACAACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAAUGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCAAUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGUCAAGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUCGACAGCGUCGAAAUCAGCGGAGUCGAAGACAGAUUCAACGCAAGCCUGGGAACAUACCACGACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAAGAAAACGAAGACAUCCUGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGAGAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCGACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUCAGAGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCCACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCACAGGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGCAAACCUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGCAGACAGUCAAGGUCGUCGACGAACUGGUCAAGGUCAUGGGAAGACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAAUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGCCAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAGAACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAAGAGACAUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGCGACUACGACGUCGACGCAAUCGUCCCGCAGAGCUUCCUGAAGGACGACAGCAUCGACAACAAGGUCCUGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGUCGUCAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCAAAGCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAGGCAGAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAUCAAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAGCACGUCGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUACGACGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACACUGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAGUUCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACACGACGCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAGCUGGAAAGCGAAUUCGUCUACGGAGACUACAAGGUCUACGACGUCAGAAAGAUGAUCGCAAAGAGCGAACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACAGCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAACGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGGAGAAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGACUUCGCAACAGUCAGAAAGGUCCUGAGCAUGCCGCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGCAAGGAAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGAAAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAGUCGCAUACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAACUGCUGGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUCAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUCGAACUGGAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCAGGAGAACUGCAGAAGGGAAACGAACUGGCACUGCCGAGCAAGUACGUCAACUUCCUGUACCUGGCAAGCCACUACGAAAAGCUGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUCGUCGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACAGAUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAAACCUGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACAAGCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUGUUCACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUACUUCGACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAGGAAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACAGGACUGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAGACGGAAGCGGAAGCCCGAAGAAGAAGAGAAAGGUCGACGGAAGCCCGAAGAAGAAGAGAAAGGUCGACAGCGGA T7 Promoter TAATACGACTCACTATA231 Human beta- ACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACC 232globin 5′ UTR Human beta-GCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAG 233 globin 3′TCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTC UTRTGCCTAATAAAAAACATTTATTTTCATTGC Human alpha-CATAAACCCTGGCGCGCTCGCGGCCCGGCACTCTTCTGGTCCCCACAGACT 234 globin 5′CAGAGAGAACCCACC UTR Human alpha-GCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCC 235 globin 3′CTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAG UTR TGGGCGGC XenopusAAGCTCAGAATAAACGCTCAACTTTGGCC 236 laevis beta- globin 5′ UTR XenopusACCAGCCTCAAGAACACCCGAATGGAGTCTCTAAGCTACATAATACCAACT 237 laevis beta-TACACTTTACAAAATGTTGTCCCCCAAAATGTAGCCATTCGTATCTGCTCC globin 3′TAATAAAAAGAAAGTTTCTTCACATTCT UTR Bovine CAGGGTCCTGTGGACAGCTCACCAGCT 238Growth Hormone 5′ UTR BovineTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGA 239 GrowthAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCA Hormone 3′ UTRMus musculus GCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTG 240hemoglobin CACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAG alpha, adultchain 1 (Hba-a1), 3′ UTR HSD17B4 5′TCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGTGTGTCGTTGCA 241 UTR GGCCTTATTCG282 single mU*mU*mA*CAGCCACGUCUACAGCAGUUUUAGAmGmCmUmAmGmAmAmAm 242guide RNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA targetingmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU the mouse TTR geneNot used 243 Cas9 GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGTGTGTCGTT244 transcript GCAGGCCTTATTCGGATCCATGGACAAGAAGTACAGCATCGGACTGGACATwith 5′ UTR CGGAACAAACAGCGTCGGATGGGCAGTCATCACAGACGAATACAAGGTCCCof HSD, ORF GAGCAAGAAGTTCAAGGTCCTGGGAAACACAGACAGACACAGCATCAAGAAcorresponding GAACCTGATCGGAGCACTGCTGTTCGACAGCGGAGAAACAGCAGAAGCAACto SEQ ID AAGACTGAAGAGAACAGCAAGAAGAAGATACACAAGAAGAAAGAACAGAATNO: 204, and CTGCTACCTGCAGGAAATCTTCAGCAACGAAATGGCAAAGGTCGACGACAG3′ UTR of CTTCTTCCACAGACTGGAAGAAAGCTTCCTGGTCGAAGAAGACAAGAAGCA ALBCGAAAGACACCCGATCTTCGGAAACATCGTCGACGAAGTCGCATACCACGAAAAGTACCCGACAATCTACCACCTGAGAAAGAAGCTGGTCGACAGCACAGACAAGGCAGACCTGAGACTGATCTACCTGGCACTGGCACACATGATCAAGTTCAGAGGACACTTCCTGATCGAAGGAGACCTGAACCCGGACAACAGCGACGTCGACAAGCTGTTCATCCAGCTGGTCCAGACATACAACCAGCTGTTCGAAGAAAACCCGATCAACGCAAGCGGAGTCGACGCAAAGGCAATCCTGAGCGCAAGACTGAGCAAGAGCAGAAGACTGGAAAACCTGATCGCACAGCTGCCGGGAGAAAAGAAGAACGGACTGTTCGGAAACCTGATCGCACTGAGCCTGGGACTGACACCGAACTTCAAGAGCAACTTCGACCTGGCAGAAGACGCAAAGCTGCAGCTGAGCAAGGACACATACGACGACGACCTGGACAACCTGCTGGCACAGATCGGAGACCAGTACGCAGACCTGTTCCTGGCAGCAAAGAACCTGAGCGACGCAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAAATCACAAAGGCACCGCTGAGCGCAAGCATGATCAAGAGATACGACGAACACCACCAGGACCTGACACTGCTGAAGGCACTGGTCAGACAGCAGCTGCCGGAAAAGTACAAGGAAATCTTCTTCGACCAGAGCAAGAACGGATACGCAGGATACATCGACGGAGGAGCAAGCCAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAAGATGGACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAAGACCTGCTGAGAAAGCAGAGAACATTCGACAACGGAAGCATCCCGCACCAGATCCACCTGGGAGAACTGCACGCAATCCTGAGAAGACAGGAAGACTTCTACCCGTTCCTGAAGGACAACAGAGAAAAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTACGTCGGACCGCTGGCAAGAGGAAACAGCAGATTCGCATGGATGACAAGAAAGAGCGAAGAAACAATCACACCGTGGAACTTCGAAGAAGTCGTCGACAAGGGAGCAAGCGCACAGAGCTTCATCGAAAGAATGACAAACTTCGACAAGAACCTGCCGAACGAAAAGGTCCTGCCGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTACAACGAACTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCCGGCATTCCTGAGCGGAGAACAGAAGAAGGCAATCGTCGACCTGCTGTTCAAGACAAACAGAAAGGTCACAGTCAAGCAGCTGAAGGAAGACTACTTCAAGAAGATCGAATGCTTCGACAGCGTCGAAATCAGCGGAGTCGAAGACAGATTCAACGCAAGCCTGGGAACATACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGACAACGAAGAAAACGAAGACATCCTGGAAGACATCGTCCTGACACTGACACTGTTCGAAGACAGAGAAATGATCGAAGAAAGACTGAAGACATACGCACACCTGTTCGACGACAAGGTCATGAAGCAGCTGAAGAGAAGAAGATACACAGGATGGGGAAGACTGAGCAGAAAGCTGATCAACGGAATCAGAGACAAGCAGAGCGGAAAGACAATCCTGGACTTCCTGAAGAGCGACGGATTCGCAAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACATTCAAGGAAGACATCCAGAAGGCACAGGTCAGCGGACAGGGAGACAGCCTGCACGAACACATCGCAAACCTGGCAGGAAGCCCGGCAATCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAACTGGTCAAGGTCATGGGAAGACACAAGCCGGAAAACATCGTCATCGAAATGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAATGAAGAGAATCGAAGAAGGAATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACACCCGGTCGAAAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCTGCAGAACGGAAGAGACATGTACGTCGACCAGGAACTGGACATCAACAGACTGAGCGACTACGACGTCGACCACATCGTCCCGCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTCCTGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGTCCCGAGCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGACAGCTGCTGAACGCAAAGCTGATCACACAGAGAAAGTTCGACAACCTGACAAAGGCAGAGAGAGGAGGACTGAGCGAACTGGACAAGGCAGGATTCATCAAGAGACAGCTGGTCGAAACAAGACAGATCACAAAGCACGTCGCACAGATCCTGGACAGCAGAATGAACACAAAGTACGACGAAAACGACAAGCTGATCAGAGAAGTCAAGGTCATCACACTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGACTTCCAGTTCTACAAGGTCAGAGAAATCAACAACTACCACCACGCACACGACGCATACCTGAACGCAGTCGTCGGAACAGCACTGATCAAGAAGTACCCGAAGCTGGAAAGCGAATTCGTCTACGGAGACTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGAGCGAACAGGAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAAGACAGAAATCACACTGGCAAACGGAGAAATCAGAAAGAGACCGCTGATCGAAACAAACGGAGAAACAGGAGAAATCGTCTGGGACAAGGGAAGAGACTTCGCAACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCGTCAAGAAGACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAGCATCCTGCCGAAGAGAAACAGCGACAAGCTGATCGCAAGAAAGAAGGACTGGGACCCGAAGAAGTACGGAGGATTCGACAGCCCGACAGTCGCATACAGCGTCCTGGTCGTCGCAAAGGTCGAAAAGGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGCTGGGAATCACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGATCGACTTCCTGGAAGCAAAGGGATACAAGGAAGTCAAGAAGGACCTGATCATCAAGCTGCCGAAGTACAGCCTGTTCGAACTGGAAAACGGAAGAAAGAGAATGCTGGCAAGCGCAGGAGAACTGCAGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGTCAACTTCCTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCTGTTCGTCGAACAGCACAAGCACTACCTGGACGAAATCATCGAACAGATCAGCGAATTCAGCAAGAGAGTCATCCTGGCAGACGCAAACCTGGACAAGGTCCTGAGCGCATACAACAAGCACAGAGACAAGCCGATCAGAGAACAGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTGGGAGCACCGGCAGCATTCAAGTACTTCGACACAACAATCGACAGAAAGAGATACACAAGCACAAAGGAAGTCCTGGACGCAACACTGATCCACCAGAGCATCACAGGACTGTACGAAACAAGAATCGACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAAGCCCGAAGAAGAAGAGAAAGGTCTAGCTAGCCATCACATTTAAAAGCATCTCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAATAGCTTATTCATCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAACATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAATAAA AAATGGAAAGAACCTCGAGAlternative ATGGATAAGAAGTACTCGATCGGGCTGGATATCGGAACTAATTCCGTGGGT 245Cas9 ORF TGGGCAGTGATCACGGATGAATACAAAGTGCCGTCCAAGAAGTTCAAGGTC with 19.36%CTGGGGAACACCGATAGACACAGCATCAAGAAGAATCTCATCGGAGCCCTG U contentCTGTTTGACTCCGGCGAAACCGCAGAAGCGACCCGGCTCAAACGTACCGCGAGGCGACGCTACACCCGGCGGAAGAATCGCATCTGCTATCTGCAAGAAATCTTTTCGAACGAAATGGCAAAGGTGGACGACAGCTTCTTCCACCGCCTGGAAGAATCTTTCCTGGTGGAGGAGGACAAGAAGCATGAACGGCATCCTATCTTTGGAAACATCGTGGACGAAGTGGCGTACCACGAAAAGTACCCGACCATCTACCATCTGCGGAAGAAGTTGGTTGACTCAACTGACAAGGCCGACCTCAGATTGATCTACTTGGCCCTCGCCCATATGATCAAATTCCGCGGACACTTCCTGATCGAAGGCGATCTGAACCCTGATAACTCCGACGTGGATAAGCTGTTCATTCAACTGGTGCAGACCTACAACCAACTGTTCGAAGAAAACCCAATCAATGCCAGCGGCGTCGATGCCAAGGCCATCCTGTCCGCCCGGCTGTCGAAGTCGCGGCGCCTCGAAAACCTGATCGCACAGCTGCCGGGAGAGAAGAAGAACGGACTTTTCGGCAACTTGATCGCTCTCTCACTGGGACTCACTCCCAATTTCAAGTCCAATTTTGACCTGGCCGAGGACGCGAAGCTGCAACTCTCAAAGGACACCTACGACGACGACTTGGACAATTTGCTGGCACAAATTGGCGATCAGTACGCGGATCTGTTCCTTGCCGCTAAGAACCTTTCGGACGCAATCTTGCTGTCCGATATCCTGCGCGTGAACACCGAAATAACCAAAGCGCCGCTTAGCGCCTCGATGATTAAGCGGTACGACGAGCATCACCAGGATCTCACGCTGCTCAAAGCGCTCGTGAGACAGCAACTGCCTGAAAAGTACAAGGAGATTTTCTTCGACCAGTCCAAGAATGGGTACGCAGGGTACATCGATGGAGGCGCCAGCCAGGAAGAGTTCTATAAGTTCATCAAGCCAATCCTGGAAAAGATGGACGGAACCGAAGAACTGCTGGTCAAGCTGAACAGGGAGGATCTGCTCCGCAAACAGAGAACCTTTGACAACGGAAGCATTCCACACCAGATCCATCTGGGTGAGCTGCACGCCATCTTGCGGCGCCAGGAGGACTTTTACCCATTCCTCAAGGACAACCGGGAAAAGATCGAGAAAATTCTGACGTTCCGCATCCCGTATTACGTGGGCCCACTGGCGCGCGGCAATTCGCGCTTCGCGTGGATGACTAGAAAATCAGAGGAAACCATCACTCCTTGGAATTTCGAGGAAGTTGTGGATAAGGGAGCTTCGGCACAATCCTTCATCGAACGAATGACCAACTTCGACAAGAATCTCCCAAACGAGAAGGTGCTTCCTAAGCACAGCCTCCTTTACGAATACTTCACTGTCTACAACGAACTGACTAAAGTGAAATACGTTACTGAAGGAATGAGGAAGCCGGCCTTTCTGAGCGGAGAACAGAAGAAAGCGATTGTCGATCTGCTGTTCAAGACCAACCGCAAGGTGACCGTCAAGCAGCTTAAAGAGGACTACTTCAAGAAGATCGAGTGTTTCGACTCAGTGGAAATCAGCGGAGTGGAGGACAGATTCAACGCTTCGCTGGGAACCTATCATGATCTCCTGAAGATCATCAAGGACAAGGACTTCCTTGACAACGAGGAGAACGAGGACATCCTGGAAGATATCGTCCTGACCTTGACCCTTTTCGAGGATCGCGAGATGATCGAGGAGAGGCTTAAGACCTACGCTCATCTCTTCGACGATAAGGTCATGAAACAACTCAAGCGCCGCCGGTACACTGGTTGGGGCCGCCTCTCCCGCAAGCTGATCAACGGTATTCGCGATAAACAGAGCGGTAAAACTATCCTGGATTTCCTCAAATCGGATGGCTTCGCTAATCGTAACTTCATGCAGTTGATCCACGACGACAGCCTGACCTTTAAGGAGGACATCCAGAAAGCACAAGTGAGCGGACAGGGAGACTCACTCCATGAACACATCGCGAATCTGGCCGGTTCGCCGGCGATTAAGAAGGGAATCCTGCAAACTGTGAAGGTGGTGGACGAGCTGGTGAAGGTCATGGGACGGCACAAACCGGAGAATATCGTGATTGAAATGGCCCGAGAAAACCAGACTACCCAGAAGGGCCAGAAGAACTCCCGCGAAAGGATGAAGCGGATCGAAGAAGGAATCAAGGAGCTGGGCAGCCAGATCCTGAAAGAGCACCCGGTGGAAAACACGCAGCTGCAGAACGAGAAGCTCTACCTGTACTATTTGCAAAATGGACGGGACATGTACGTGGACCAAGAGCTGGACATCAATCGGTTGTCTGATTACGACGTGGACCACATCGTTCCACAGTCCTTTCTGAAGGATGACTCCATCGATAACAAGGTGTTGACTCGCAGCGACAAGAACAGAGGGAAGTCAGATAATGTGCCATCGGAGGAGGTCGTGAAGAAGATGAAGAATTACTGGCGGCAGCTCCTGAATGCGAAGCTGATTACCCAGAGAAAGTTTGACAATCTCACTAAAGCCGAGCGCGGCGGACTCTCAGAGCTGGATAAGGCTGGATTCATCAAACGGCAGCTGGTCGAGACTCGGCAGATTACCAAGCACGTGGCGCAGATCCTGGACTCCCGCATGAACACTAAATACGACGAGAACGATAAGCTCATCCGGGAAGTGAAGGTGATTACCCTGAAAAGCAAACTTGTGTCGGACTTTCGGAAGGACTTTCAGTTTTACAAAGTGAGAGAAATCAACAACTACCATCACGCGCATGACGCATACCTCAACGCTGTGGTCGGCACCGCCCTGATCAAGAAGTACCCTAAACTTGAATCGGAGTTTGTGTACGGAGACTACAAGGTCTACGACGTGAGGAAGATGATAGCCAAGTCCGAACAGGAAATCGGGAAAGCAACTGCGAAATACTTCTTTTACTCAAACATCATGAACTTCTTCAAGACTGAAATTACGCTGGCCAATGGAGAAATCAGGAAGAGGCCACTGATCGAAACTAACGGAGAAACGGGCGAAATCGTGTGGGACAAGGGCAGGGACTTCGCAACTGTTCGCAAAGTGCTCTCTATGCCGCAAGTCAATATTGTGAAGAAAACCGAAGTGCAAACCGGCGGATTTTCAAAGGAATCGATCCTCCCAAAGAGAAATAGCGACAAGCTCATTGCACGCAAGAAAGACTGGGACCCGAAGAAGTACGGAGGATTCGATTCGCCGACTGTCGCATACTCCGTCCTCGTGGTGGCCAAGGTGGAGAAGGGAAAGAGCAAGAAGCTCAAATCCGTCAAAGAGCTGCTGGGGATTACCATCATGGAACGATCCTCGTTCGAGAAGAACCCGATTGATTTCCTGGAGGCGAAGGGTTACAAGGAGGTGAAGAAGGATCTGATCATCAAACTGCCCAAGTACTCACTGTTCGAACTGGAAAATGGTCGGAAGCGCATGCTGGCTTCGGCCGGAGAACTCCAGAAAGGAAATGAGCTGGCCTTGCCTAGCAAGTACGTCAACTTCCTCTATCTTGCTTCGCACTACGAGAAACTCAAAGGGTCACCGGAAGATAACGAACAGAAGCAGCTTTTCGTGGAGCAGCACAAGCATTATCTGGATGAAATCATCGAACAAATCTCCGAGTTTTCAAAGCGCGTGATCCTCGCCGACGCCAACCTCGACAAAGTCCTGTCGGCCTACAATAAGCATAGAGATAAGCCGATCAGAGAACAGGCCGAGAACATTATCCACTTGTTCACCCTGACTAACCTGGGAGCTCCAGCCGCCTTCAAGTACTTCGATACTACTATCGACCGCAAAAGATACACGTCCACCAAGGAAGTTCTGGACGCGACCCTGATCCACCAAAGCATCACTGGACTCTACGAAACTAGGATCGATCTGTCGCAGCTGGGTGGCGATGGTGGCGGTGGATCCTACCCATACGACGTGCCTGACTACGCCTCCGGAGGTGGTGGCCCCAAGAAGAAACGGAAGGTG TGATAG Cas9GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGTGTGTCGTT 246 transcriptGCAGGCCTTATTCGGATCTGCCACCATGGATAAGAAGTACTCGATCGGGCT with 5′ UTRGGATATCGGAACTAATTCCGTGGGTTGGGCAGTGATCACGGATGAATACAA of HSD, ORFAGTGCCGTCCAAGAAGTTCAAGGTCCTGGGGAACACCGATAGACACAGCAT correspondingCAAGAAGAATCTCATCGGAGCCCTGCTGTTTGACTCCGGCGAAACCGCAGA to SEQ IDAGCGACCCGGCTCAAACGTACCGCGAGGCGACGCTACACCCGGCGGAAGAA NO: 245,TCGCATCTGCTATCTGCAAGAAATCTTTTCGAACGAAATGGCAAAGGTGGA KozakCGACAGCTTCTTCCACCGCCTGGAAGAATCTTTCCTGGTGGAGGAGGACAA sequence,GAAGCATGAACGGCATCCTATCTTTGGAAACATCGTGGACGAAGTGGCGTA and 3′ UTRCCACGAAAAGTACCCGACCATCTACCATCTGCGGAAGAAGTTGGTTGACTC of ALBAACTGACAAGGCCGACCTCAGATTGATCTACTTGGCCCTCGCCCATATGATCAAATTCCGCGGACACTTCCTGATCGAAGGCGATCTGAACCCTGATAACTCCGACGTGGATAAGCTGTTCATTCAACTGGTGCAGACCTACAACCAACTGTTCGAAGAAAACCCAATCAATGCCAGCGGCGTCGATGCCAAGGCCATCCTGTCCGCCCGGCTGTCGAAGTCGCGGCGCCTCGAAAACCTGATCGCACAGCTGCCGGGAGAGAAGAAGAACGGACTTTTCGGCAACTTGATCGCTCTCTCACTGGGACTCACTCCCAATTTCAAGTCCAATTTTGACCTGGCCGAGGACGCGAAGCTGCAACTCTCAAAGGACACCTACGACGACGACTTGGACAATTTGCTGGCACAAATTGGCGATCAGTACGCGGATCTGTTCCTTGCCGCTAAGAACCTTTCGGACGCAATCTTGCTGTCCGATATCCTGCGCGTGAACACCGAAATAACCAAAGCGCCGCTTAGCGCCTCGATGATTAAGCGGTACGACGAGCATCACCAGGATCTCACGCTGCTCAAAGCGCTCGTGAGACAGCAACTGCCTGAAAAGTACAAGGAGATTTTCTTCGACCAGTCCAAGAATGGGTACGCAGGGTACATCGATGGAGGCGCCAGCCAGGAAGAGTTCTATAAGTTCATCAAGCCAATCCTGGAAAAGATGGACGGAACCGAAGAACTGCTGGTCAAGCTGAACAGGGAGGATCTGCTCCGCAAACAGAGAACCTTTGACAACGGAAGCATTCCACACCAGATCCATCTGGGTGAGCTGCACGCCATCTTGCGGCGCCAGGAGGACTTTTACCCATTCCTCAAGGACAACCGGGAAAAGATCGAGAAAATTCTGACGTTCCGCATCCCGTATTACGTGGGCCCACTGGCGCGCGGCAATTCGCGCTTCGCGTGGATGACTAGAAAATCAGAGGAAACCATCACTCCTTGGAATTTCGAGGAAGTTGTGGATAAGGGAGCTTCGGCACAATCCTTCATCGAACGAATGACCAACTTCGACAAGAATCTCCCAAACGAGAAGGTGCTTCCTAAGCACAGCCTCCTTTACGAATACTTCACTGTCTACAACGAACTGACTAAAGTGAAATACGTTACTGAAGGAATGAGGAAGCCGGCCTTTCTGAGCGGAGAACAGAAGAAAGCGATTGTCGATCTGCTGTTCAAGACCAACCGCAAGGTGACCGTCAAGCAGCTTAAAGAGGACTACTTCAAGAAGATCGAGTGTTTCGACTCAGTGGAAATCAGCGGAGTGGAGGACAGATTCAACGCTTCGCTGGGAACCTATCATGATCTCCTGAAGATCATCAAGGACAAGGACTTCCTTGACAACGAGGAGAACGAGGACATCCTGGAAGATATCGTCCTGACCTTGACCCTTTTCGAGGATCGCGAGATGATCGAGGAGAGGCTTAAGACCTACGCTCATCTCTTCGACGATAAGGTCATGAAACAACTCAAGCGCCGCCGGTACACTGGTTGGGGCCGCCTCTCCCGCAAGCTGATCAACGGTATTCGCGATAAACAGAGCGGTAAAACTATCCTGGATTTCCTCAAATCGGATGGCTTCGCTAATCGTAACTTCATGCAGTTGATCCACGACGACAGCCTGACCTTTAAGGAGGACATCCAGAAAGCACAAGTGAGCGGACAGGGAGACTCACTCCATGAACACATCGCGAATCTGGCCGGTTCGCCGGCGATTAAGAAGGGAATCCTGCAAACTGTGAAGGTGGTGGACGAGCTGGTGAAGGTCATGGGACGGCACAAACCGGAGAATATCGTGATTGAAATGGCCCGAGAAAACCAGACTACCCAGAAGGGCCAGAAGAACTCCCGCGAAAGGATGAAGCGGATCGAAGAAGGAATCAAGGAGCTGGGCAGCCAGATCCTGAAAGAGCACCCGGTGGAAAACACGCAGCTGCAGAACGAGAAGCTCTACCTGTACTATTTGCAAAATGGACGGGACATGTACGTGGACCAAGAGCTGGACATCAATCGGTTGTCTGATTACGACGTGGACCACATCGTTCCACAGTCCTTTCTGAAGGATGACTCCATCGATAACAAGGTGTTGACTCGCAGCGACAAGAACAGAGGGAAGTCAGATAATGTGCCATCGGAGGAGGTCGTGAAGAAGATGAAGAATTACTGGCGGCAGCTCCTGAATGCGAAGCTGATTACCCAGAGAAAGTTTGACAATCTCACTAAAGCCGAGCGCGGCGGACTCTCAGAGCTGGATAAGGCTGGATTCATCAAACGGCAGCTGGTCGAGACTCGGCAGATTACCAAGCACGTGGCGCAGATCCTGGACTCCCGCATGAACACTAAATACGACGAGAACGATAAGCTCATCCGGGAAGTGAAGGTGATTACCCTGAAAAGCAAACTTGTGTCGGACTTTCGGAAGGACTTTCAGTTTTACAAAGTGAGAGAAATCAACAACTACCATCACGCGCATGACGCATACCTCAACGCTGTGGTCGGCACCGCCCTGATCAAGAAGTACCCTAAACTTGAATCGGAGTTTGTGTACGGAGACTACAAGGTCTACGACGTGAGGAAGATGATAGCCAAGTCCGAACAGGAAATCGGGAAAGCAACTGCGAAATACTTCTTTTACTCAAACATCATGAACTTCTTCAAGACTGAAATTACGCTGGCCAATGGAGAAATCAGGAAGAGGCCACTGATCGAAACTAACGGAGAAACGGGCGAAATCGTGTGGGACAAGGGCAGGGACTTCGCAACTGTTCGCAAAGTGCTCTCTATGCCGCAAGTCAATATTGTGAAGAAAACCGAAGTGCAAACCGGCGGATTTTCAAAGGAATCGATCCTCCCAAAGAGAAATAGCGACAAGCTCATTGCACGCAAGAAAGACTGGGACCCGAAGAAGTACGGAGGATTCGATTCGCCGACTGTCGCATACTCCGTCCTCGTGGTGGCCAAGGTGGAGAAGGGAAAGAGCAAGAAGCTCAAATCCGTCAAAGAGCTGCTGGGGATTACCATCATGGAACGATCCTCGTTCGAGAAGAACCCGATTGATTTCCTGGAGGCGAAGGGTTACAAGGAGGTGAAGAAGGATCTGATCATCAAACTGCCCAAGTACTCACTGTTCGAACTGGAAAATGGTCGGAAGCGCATGCTGGCTTCGGCCGGAGAACTCCAGAAAGGAAATGAGCTGGCCTTGCCTAGCAAGTACGTCAACTTCCTCTATCTTGCTTCGCACTACGAGAAACTCAAAGGGTCACCGGAAGATAACGAACAGAAGCAGCTTTTCGTGGAGCAGCACAAGCATTATCTGGATGAAATCATCGAACAAATCTCCGAGTTTTCAAAGCGCGTGATCCTCGCCGACGCCAACCTCGACAAAGTCCTGTCGGCCTACAATAAGCATAGAGATAAGCCGATCAGAGAACAGGCCGAGAACATTATCCACTTGTTCACCCTGACTAACCTGGGAGCTCCAGCCGCCTTCAAGTACTTCGATACTACTATCGACCGCAAAAGATACACGTCCACCAAGGAAGTTCTGGACGCGACCCTGATCCACCAAAGCATCACTGGACTCTACGAAACTAGGATCGATCTGTCGCAGCTGGGTGGCGATGGTGGCGGTGGATCCTACCCATACGACGTGCCTGACTACGCCTCCGGAGGTGGTGGCCCCAAGAAGAAACGGAAGGTGTGATAGCTAGCCATCACATTTAAAAGCATCTCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAATAGCTTATTCATCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAACATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAATAAAAAATGGAAAGAACCTCGAG Cas9GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGTGTGTCGTT 247 transcriptGCAGGCCTTATTCGGATCTATGGATAAGAAGTACTCGATCGGGCTGGATAT with 5′ UTRCGGAACTAATTCCGTGGGTTGGGCAGTGATCACGGATGAATACAAAGTGCC of HSD, ORFGTCCAAGAAGTTCAAGGTCCTGGGGAACACCGATAGACACAGCATCAAGAA correspondingGAATCTCATCGGAGCCCTGCTGTTTGACTCCGGCGAAACCGCAGAAGCGAC to SEQ IDCCGGCTCAAACGTACCGCGAGGCGACGCTACACCCGGCGGAAGAATCGCAT NO: 245, andCTGCTATCTGCAAGAAATCTTTTCGAACGAAATGGCAAAGGTGGACGACAG 3′ UTR ofCTTCTTCCACCGCCTGGAAGAATCTTTCCTGGTGGAGGAGGACAAGAAGCA ALBTGAACGGCATCCTATCTTTGGAAACATCGTGGACGAAGTGGCGTACCACGAAAAGTACCCGACCATCTACCATCTGCGGAAGAAGTTGGTTGACTCAACTGACAAGGCCGACCTCAGATTGATCTACTTGGCCCTCGCCCATATGATCAAATTCCGCGGACACTTCCTGATCGAAGGCGATCTGAACCCTGATAACTCCGACGTGGATAAGCTGTTCATTCAACTGGTGCAGACCTACAACCAACTGTTCGAAGAAAACCCAATCAATGCCAGCGGCGTCGATGCCAAGGCCATCCTGTCCGCCCGGCTGTCGAAGTCGCGGCGCCTCGAAAACCTGATCGCACAGCTGCCGGGAGAGAAGAAGAACGGACTTTTCGGCAACTTGATCGCTCTCTCACTGGGACTCACTCCCAATTTCAAGTCCAATTTTGACCTGGCCGAGGACGCGAAGCTGCAACTCTCAAAGGACACCTACGACGACGACTTGGACAATTTGCTGGCACAAATTGGCGATCAGTACGCGGATCTGTTCCTTGCCGCTAAGAACCTTTCGGACGCAATCTTGCTGTCCGATATCCTGCGCGTGAACACCGAAATAACCAAAGCGCCGCTTAGCGCCTCGATGATTAAGCGGTACGACGAGCATCACCAGGATCTCACGCTGCTCAAAGCGCTCGTGAGACAGCAACTGCCTGAAAAGTACAAGGAGATTTTCTTCGACCAGTCCAAGAATGGGTACGCAGGGTACATCGATGGAGGCGCCAGCCAGGAAGAGTTCTATAAGTTCATCAAGCCAATCCTGGAAAAGATGGACGGAACCGAAGAACTGCTGGTCAAGCTGAACAGGGAGGATCTGCTCCGCAAACAGAGAACCTTTGACAACGGAAGCATTCCACACCAGATCCATCTGGGTGAGCTGCACGCCATCTTGCGGCGCCAGGAGGACTTTTACCCATTCCTCAAGGACAACCGGGAAAAGATCGAGAAAATTCTGACGTTCCGCATCCCGTATTACGTGGGCCCACTGGCGCGCGGCAATTCGCGCTTCGCGTGGATGACTAGAAAATCAGAGGAAACCATCACTCCTTGGAATTTCGAGGAAGTTGTGGATAAGGGAGCTTCGGCACAATCCTTCATCGAACGAATGACCAACTTCGACAAGAATCTCCCAAACGAGAAGGTGCTTCCTAAGCACAGCCTCCTTTACGAATACTTCACTGTCTACAACGAACTGACTAAAGTGAAATACGTTACTGAAGGAATGAGGAAGCCGGCCTTTCTGAGCGGAGAACAGAAGAAAGCGATTGTCGATCTGCTGTTCAAGACCAACCGCAAGGTGACCGTCAAGCAGCTTAAAGAGGACTACTTCAAGAAGATCGAGTGTTTCGACTCAGTGGAAATCAGCGGAGTGGAGGACAGATTCAACGCTTCGCTGGGAACCTATCATGATCTCCTGAAGATCATCAAGGACAAGGACTTCCTTGACAACGAGGAGAACGAGGACATCCTGGAAGATATCGTCCTGACCTTGACCCTTTTCGAGGATCGCGAGATGATCGAGGAGAGGCTTAAGACCTACGCTCATCTCTTCGACGATAAGGTCATGAAACAACTCAAGCGCCGCCGGTACACTGGTTGGGGCCGCCTCTCCCGCAAGCTGATCAACGGTATTCGCGATAAACAGAGCGGTAAAACTATCCTGGATTTCCTCAAATCGGATGGCTTCGCTAATCGTAACTTCATGCAGTTGATCCACGACGACAGCCTGACCTTTAAGGAGGACATCCAGAAAGCACAAGTGAGCGGACAGGGAGACTCACTCCATGAACACATCGCGAATCTGGCCGGTTCGCCGGCGATTAAGAAGGGAATCCTGCAAACTGTGAAGGTGGTGGACGAGCTGGTGAAGGTCATGGGACGGCACAAACCGGAGAATATCGTGATTGAAATGGCCCGAGAAAACCAGACTACCCAGAAGGGCCAGAAGAACTCCCGCGAAAGGATGAAGCGGATCGAAGAAGGAATCAAGGAGCTGGGCAGCCAGATCCTGAAAGAGCACCCGGTGGAAAACACGCAGCTGCAGAACGAGAAGCTCTACCTGTACTATTTGCAAAATGGACGGGACATGTACGTGGACCAAGAGCTGGACATCAATCGGTTGTCTGATTACGACGTGGACCACATCGTTCCACAGTCCTTTCTGAAGGATGACTCCATCGATAACAAGGTGTTGACTCGCAGCGACAAGAACAGAGGGAAGTCAGATAATGTGCCATCGGAGGAGGTCGTGAAGAAGATGAAGAATTACTGGCGGCAGCTCCTGAATGCGAAGCTGATTACCCAGAGAAAGTTTGACAATCTCACTAAAGCCGAGCGCGGCGGACTCTCAGAGCTGGATAAGGCTGGATTCATCAAACGGCAGCTGGTCGAGACTCGGCAGATTACCAAGCACGTGGCGCAGATCCTGGACTCCCGCATGAACACTAAATACGACGAGAACGATAAGCTCATCCGGGAAGTGAAGGTGATTACCCTGAAAAGCAAACTTGTGTCGGACTTTCGGAAGGACTTTCAGTTTTACAAAGTGAGAGAAATCAACAACTACCATCACGCGCATGACGCATACCTCAACGCTGTGGTCGGCACCGCCCTGATCAAGAAGTACCCTAAACTTGAATCGGAGTTTGTGTACGGAGACTACAAGGTCTACGACGTGAGGAAGATGATAGCCAAGTCCGAACAGGAAATCGGGAAAGCAACTGCGAAATACTTCTTTTACTCAAACATCATGAACTTCTTCAAGACTGAAATTACGCTGGCCAATGGAGAAATCAGGAAGAGGCCACTGATCGAAACTAACGGAGAAACGGGCGAAATCGTGTGGGACAAGGGCAGGGACTTCGCAACTGTTCGCAAAGTGCTCTCTATGCCGCAAGTCAATATTGTGAAGAAAACCGAAGTGCAAACCGGCGGATTTTCAAAGGAATCGATCCTCCCAAAGAGAAATAGCGACAAGCTCATTGCACGCAAGAAAGACTGGGACCCGAAGAAGTACGGAGGATTCGATTCGCCGACTGTCGCATACTCCGTCCTCGTGGTGGCCAAGGTGGAGAAGGGAAAGAGCAAGAAGCTCAAATCCGTCAAAGAGCTGCTGGGGATTACCATCATGGAACGATCCTCGTTCGAGAAGAACCCGATTGATTTCCTGGAGGCGAAGGGTTACAAGGAGGTGAAGAAGGATCTGATCATCAAACTGCCCAAGTACTCACTGTTCGAACTGGAAAATGGTCGGAAGCGCATGCTGGCTTCGGCCGGAGAACTCCAGAAAGGAAATGAGCTGGCCTTGCCTAGCAAGTACGTCAACTTCCTCTATCTTGCTTCGCACTACGAGAAACTCAAAGGGTCACCGGAAGATAACGAACAGAAGCAGCTTTTCGTGGAGCAGCACAAGCATTATCTGGATGAAATCATCGAACAAATCTCCGAGTTTTCAAAGCGCGTGATCCTCGCCGACGCCAACCTCGACAAAGTCCTGTCGGCCTACAATAAGCATAGAGATAAGCCGATCAGAGAACAGGCCGAGAACATTATCCACTTGTTCACCCTGACTAACCTGGGAGCTCCAGCCGCCTTCAAGTACTTCGATACTACTATCGACCGCAAAAGATACACGTCCACCAAGGAAGTTCTGGACGCGACCCTGATCCACCAAAGCATCACTGGACTCTACGAAACTAGGATCGATCTGTCGCAGCTGGGTGGCGATGGTGGCGGTGGATCCTACCCATACGACGTGCCTGACTACGCCTCCGGAGGTGGTGGCCCCAAGAAGAAACGGAAGGTGTGATAGCTAGCCATCACATTTAAAAGCATCTCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAATAGCTTATTCATCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAACATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAATAAAAAA TGGAAAGAACCTCGAGNot used 248 Cas9 GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGTGTGTCGTT249 transcript GCAGGCCTTATTCGGATCCGCCACCATGCCTAAGAAAAAGCGGAAGGTCGAcomprising CGGGGATAAGAAGTACTCAATCGGGCTGGATATCGGAACTAATTCCGTGGG KozakTTGGGCAGTGATCACGGATGAATACAAAGTGCCGTCCAAGAAGTTCAAGGT sequenceCCTGGGGAACACCGATAGACACAGCATCAAGAAAAATCTCATCGGAGCCCT with Cas9GCTGTTTGACTCCGGCGAAACCGCAGAAGCGACCCGGCTCAAACGTACCGC ORF usingGAGGCGACGCTACACCCGGCGGAAGAATCGCATCTGCTATCTGCAAGAGAT codons withCTTTTCGAACGAAATGGCAAAGGTCGACGACAGCTTCTTCCACCGCCTGGA generallyAGAATCTTTCCTGGTGGAGGAGGACAAGAAGCATGAACGGCATCCTATCTT highTGGAAACATCGTCGACGAAGTGGCGTACCACGAAAAGTACCCGACCATCTA expressionCCATCTGCGGAAGAAGTTGGTTGACTCAACTGACAAGGCCGACCTCAGATT in humansGATCTACTTGGCCCTCGCCCATATGATCAAATTCCGCGGACACTTCCTGATCGAAGGCGATCTGAACCCTGATAACTCCGACGTGGATAAGCTTTTCATTCAACTGGTGCAGACCTACAACCAACTGTTCGAAGAAAACCCAATCAATGCTAGCGGCGTCGATGCCAAGGCCATCCTGTCCGCCCGGCTGTCGAAGTCGCGGCGCCTCGAAAACCTGATCGCACAGCTGCCGGGAGAGAAAAAGAACGGACTTTTCGGCAACTTGATCGCTCTCTCACTGGGACTCACTCCCAATTTCAAGTCCAATTTTGACCTGGCCGAGGACGCGAAGCTGCAACTCTCAAAGGACACCTACGACGACGACTTGGACAATTTGCTGGCACAAATTGGCGATCAGTACGCGGATCTGTTCCTTGCCGCTAAGAACCTTTCGGACGCAATCTTGCTGTCCGATATCCTGCGCGTGAACACCGAAATAACCAAAGCGCCGCTTAGCGCCTCGATGATTAAGCGGTACGACGAGCATCACCAGGATCTCACGCTGCTCAAAGCGCTCGTGAGACAGCAACTGCCTGAAAAGTACAAGGAGATCTTCTTCGACCAGTCCAAGAATGGGTACGCAGGGTACATCGATGGAGGCGCTAGCCAGGAAGAGTTCTATAAGTTCATCAAGCCAATCCTGGAAAAGATGGACGGAACCGAAGAACTGCTGGTCAAGCTGAACAGGGAGGATCTGCTCCGGAAACAGAGAACCTTTGACAACGGATCCATTCCCCACCAGATCCATCTGGGTGAGCTGCACGCCATCTTGCGGCGCCAGGAGGACTTTTACCCATTCCTCAAGGACAACCGGGAAAAGATCGAGAAAATTCTGACGTTCCGCATCCCGTATTACGTGGGCCCACTGGCGCGCGGCAATTCGCGCTTCGCGTGGATGACTAGAAAATCAGAGGAAACCATCACTCCTTGGAATTTCGAGGAAGTTGTGGATAAGGGAGCTTCGGCACAAAGCTTCATCGAACGAATGACCAACTTCGACAAGAATCTCCCAAACGAGAAGGTGCTTCCTAAGCACAGCCTCCTTTACGAATACTTCACTGTCTACAACGAACTGACTAAAGTGAAATACGTTACTGAAGGAATGAGGAAGCCGGCCTTTCTGTCCGGAGAACAGAAGAAAGCAATTGTCGATCTGCTGTTCAAGACCAACCGCAAGGTGACCGTCAAGCAGCTTAAAGAGGACTACTTCAAGAAGATCGAGTGTTTCGACTCAGTGGAAATCAGCGGGGTGGAGGACAGATTCAACGCTTCGCTGGGAACCTATCATGATCTCCTGAAGATCATCAAGGACAAGGACTTCCTTGACAACGAGGAGAACGAGGACATCCTGGAAGATATCGTCCTGACCTTGACCCTTTTCGAGGATCGCGAGATGATCGAGGAGAGGCTTAAGACCTACGCTCATCTCTTCGACGATAAGGTCATGAAACAACTCAAGCGCCGCCGGTACACTGGTTGGGGCCGCCTCTCCCGCAAGCTGATCAACGGTATTCGCGATAAACAGAGCGGTAAAACTATCCTGGATTTCCTCAAATCGGATGGCTTCGCTAATCGTAACTTCATGCAATTGATCCACGACGACAGCCTGACCTTTAAGGAGGACATCCAAAAAGCACAAGTGTCCGGACAGGGAGACTCACTCCATGAACACATCGCGAATCTGGCCGGTTCGCCGGCGATTAAGAAGGGAATTCTGCAAACTGTGAAGGTGGTCGACGAGCTGGTGAAGGTCATGGGACGGCACAAACCGGAGAATATCGTGATTGAAATGGCCCGAGAAAACCAGACTACCCAGAAGGGCCAGAAAAACTCCCGCGAAAGGATGAAGCGGATCGAAGAAGGAATCAAGGAGCTGGGCAGCCAGATCCTGAAAGAGCACCCGGTGGAAAACACGCAGCTGCAGAACGAGAAGCTCTACCTGTACTATTTGCAAAATGGACGGGACATGTACGTGGACCAAGAGCTGGACATCAATCGGTTGTCTGATTACGACGTGGACCACATCGTTCCACAGTCCTTTCTGAAGGATGACTCGATCGATAACAAGGTGTTGACTCGCAGCGACAAGAACAGAGGGAAGTCAGATAATGTGCCATCGGAGGAGGTCGTGAAGAAGATGAAGAATTACTGGCGGCAGCTCCTGAATGCGAAGCTGATTACCCAGAGAAAGTTTGACAATCTCACTAAAGCCGAGCGCGGCGGACTCTCAGAGCTGGATAAGGCTGGATTCATCAAACGGCAGCTGGTCGAGACTCGGCAGATTACCAAGCACGTGGCGCAGATCTTGGACTCCCGCATGAACACTAAATACGACGAGAACGATAAGCTCATCCGGGAAGTGAAGGTGATTACCCTGAAAAGCAAACTTGTGTCGGACTTTCGGAAGGACTTTCAGTTTTACAAAGTGAGAGAAATCAACAACTACCATCACGCGCATGACGCATACCTCAACGCTGTGGTCGGTACCGCCCTGATCAAAAAGTACCCTAAACTTGAATCGGAGTTTGTGTACGGAGACTACAAGGTCTACGACGTGAGGAAGATGATAGCCAAGTCCGAACAGGAAATCGGGAAAGCAACTGCGAAATACTTCTTTTACTCAAACATCATGAACTTTTTCAAGACTGAAATTACGCTGGCCAATGGAGAAATCAGGAAGAGGCCACTGATCGAAACTAACGGAGAAACGGGCGAAATCGTGTGGGACAAGGGCAGGGACTTCGCAACTGTTCGCAAAGTGCTCTCTATGCCGCAAGTCAATATTGTGAAGAAAACCGAAGTGCAAACCGGCGGATTTTCAAAGGAATCGATCCTCCCAAAGAGAAATAGCGACAAGCTCATTGCACGCAAGAAAGACTGGGACCCGAAGAAGTACGGAGGATTCGATTCGCCGACTGTCGCATACTCCGTCCTCGTGGTGGCCAAGGTGGAGAAGGGAAAGAGCAAAAAGCTCAAATCCGTCAAAGAGCTGCTGGGGATTACCATCATGGAACGATCCTCGTTCGAGAAGAACCCGATTGATTTCCTCGAGGCGAAGGGTTACAAGGAGGTGAAGAAGGATCTGATCATCAAACTCCCCAAGTACTCACTGTTCGAACTGGAAAATGGTCGGAAGCGCATGCTGGCTTCGGCCGGAGAACTCCAAAAAGGAAATGAGCTGGCCTTGCCTAGCAAGTACGTCAACTTCCTCTATCTTGCTTCGCACTACGAAAAACTCAAAGGGTCACCGGAAGATAACGAACAGAAGCAGCTTTTCGTGGAGCAGCACAAGCATTATCTGGATGAAATCATCGAACAAATCTCCGAGTTTTCAAAGCGCGTGATCCTCGCCGACGCCAACCTCGACAAAGTCCTGTCGGCCTACAATAAGCATAGAGATAAGCCGATCAGAGAACAGGCCGAGAACATTATCCACTTGTTCACCCTGACTAACCTGGGAGCCCCAGCCGCCTTCAAGTACTTCGATACTACTATCGATCGCAAAAGATACACGTCCACCAAGGAAGTTCTGGACGCGACCCTGATCCACCAAAGCATCACTGGACTCTACGAAACTAGGATCGATCTGTCGCAGCTGGGTGGCGATTGATAGTCTAGCCATCACATTTAAAAGCATCTCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAATAGCTTATTCATCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAACATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAATAAAAAATGGAAAGAACCTCGAG Cas9 ORFATGGACAAGAAGTACAGCATCGGACTGGACATCGGAACAAACAGCGTCGGA 250 with spliceTGGGCAGTCATCACAGACGAATACAAGGTCCCGAGCAAGAAGTTCAAGGTC junctionsCTGGGAAACACAGACAGACACAGCATCAAGAAGAACCTGATCGGAGCACTG removed;CTGTTCGACAGCGGAGAAACAGCAGAAGCAACAAGACTGAAGAGAACAGCA 12.75% UAGAAGAAGATACACAAGAAGAAAGAACAGAATCTGCTACCTGCAGGAAATC contentTTCAGCAACGAAATGGCAAAGGTCGACGACAGCTTCTTCCACcggCTGGAAGAAAGCTTCCTGGTCGAAGAAGACAAGAAGCACGAAAGACACCCGATCTTCGGAAACATCGTCGACGAAGTCGCATACCACGAAAAGTACCCGACAATCTACCACCTGAGAAAGAAGCTGGTCGACAGCACAGACAAGGCAGACCTGAGACTGATCTACCTGGCACTGGCACACATGATCAAGTTCAGAGGACACTTCCTGATCGAAGGAGACCTGAACCCGGACAACAGCGACGTCGACAAGCTGTTCATCCAGCTGGTCCAGACATACAACCAGCTGTTCGAAGAAAACCCGATCAACGCAAGCGGAGTCGACGCAAAGGCAATCCTGAGCGCAAGACTGAGCAAGAGCAGAAGACTGGAAAACCTGATCGCACAGCTGCCGGGAGAAAAGAAGAACGGACTGTTCGGAAACCTGATCGCACTGAGCCTGGGACTGACACCGAACTTCAAGAGCAACTTCGACCTGGCAGAAGACGCAAAGCTGCAGCTGAGCAAGGACACATACGACGACGACCTGGACAACCTGCTGGCACAGATCGGAGACCAGTACGCAGACCTGTTCCTGGCAGCAAAGAACCTGAGCGACGCAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAAATCACAAAGGCACCGCTGAGCGCAAGCATGATCAAGAGATACGACGAACACCACCAGGACCTGACACTGCTGAAGGCACTGGTCAGACAGCAGCTGCCGGAAAAGTACAAGGAAATCTTCTTCGACCAGAGCAAGAACGGATACGCAGGATACATCGACGGAGGAGCAAGCCAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAAGATGGACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAAGACCTGCTGAGAAAGCAGAGAACATTCGACAACGGAAGCATCCCGCACCAGATCCACCTGGGAGAACTGCACGCAATCCTGAGAAGACAGGAAGACTTCTACCCGTTCCTGAAGGACAACAGAGAAAAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTACGTCGGACCGCTGGCAAGAGGAAACAGCAGATTCGCATGGATGACAAGAAAGAGCGAAGAAACAATCACACCGTGGAACTTCGAAGAAGTCGTCGACAAGGGAGCAAGCGCACAGAGCTTCATCGAAAGAATGACAAACTTCGACAAGAACCTGCCGAACGAAAAGGTCCTGCCGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTACAACGAACTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCCGGCATTCCTGAGCGGAGAACAGAAGAAGGCAATCGTCGACCTGCTGTTCAAGACAAACAGAAAGGTCACAGTCAAGCAGCTGAAGGAAGACTACTTCAAGAAGATCGAATGCTTCGACAGCGTCGAAATCAGCGGAGTCGAAGACAGATTCAACGCAAGCCTGGGAACATACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGACAACGAAGAAAACGAAGACATCCTGGAAGACATCGTCCTGACACTGACACTGTTCGAAGACAGAGAAATGATCGAAGAAAGACTGAAGACATACGCACACCTGTTCGACGACAAGGTCATGAAGCAGCTGAAGAGAAGAAGATACACAGGATGGGGAAGACTGAGCAGAAAGCTGATCAACGGAATCAGAGACAAGCAGAGCGGAAAGACAATCCTGGACTTCCTGAAGAGCGACGGATTCGCAAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACATTCAAGGAAGACATCCAGAAGGCACAGGTCAGCGGACAGGGAGACAGCCTGCACGAACACATCGCAAACCTGGCAGGAAGCCCGGCAATCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAACTGGTCAAGGTCATGGGAAGACACAAGCCGGAAAACATCGTCATCGAAATGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAATGAAGAGAATCGAAGAAGGAATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACACCCGGTCGAAAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCTGCAaAACGGAAGAGACATGTACGTCGACCAGGAACTGGACATCAACAGACTGAGCGACTACGACGTCGACCACATCGTCCCGCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTCCTGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGTCCCGAGCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGACAGCTGCTGAACGCAAAGCTGATCACACAGAGAAAGTTCGACAACCTGACAAAGGCAGAGAGAGGAGGACTGAGCGAACTGGACAAGGCAGGATTCATCAAGAGACAGCTGGTCGAAACAAGACAGATCACAAAGCACGTCGCACAGATCCTGGACAGCAGAATGAACACAAAGTACGACGAAAACGACAAGCTGATCAGAGAAGTCAAGGTCATCACACTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGACTTCCAGTTCTACAAGGTCAGAGAAATCAACAACTACCACCACGCACACGACGCATACCTGAACGCAGTCGTCGGAACAGCACTGATCAAGAAGTACCCGAAGCTGGAAAGCGAATTCGTCTACGGAGACTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGAGCGAACAGGAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAAGACAGAAATCACACTGGCAAACGGAGAAATCAGAAAGAGACCGCTGATCGAAACAAACGGAGAAACAGGAGAAATCGTCTGGGACAAGGGAAGAGACTTCGCAACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCGTCAAGAAGACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAGCATCCTGCCGAAGAGAAACAGCGACAAGCTGATCGCAAGAAAGAAGGACTGGGACCCGAAGAAGTACGGAGGATTCGACAGCCCGACAGTCGCATACAGCGTCCTGGTCGTCGCAAAGGTCGAAAAGGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGCTGGGAATCACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGATCGACTTCCTGGAAGCAAAGGGATACAAGGAAGTCAAGAAGGACCTGATCATCAAGCTGCCGAAGTACAGCCTGTTCGAACTGGAAAACGGAAGAAAGAGAATGCTGGCAAGCGCAGGAGAACTGCAGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGTCAACTTCCTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCTGTTCGTCGAACAGCACAAGCACTACCTGGACGAAATCATCGAACAGATCAGCGAATTCAGCAAGAGAGTCATCCTGGCAGACGCAAACCTGGACAAGGTCCTGAGCGCATACAACAAGCACAGAGACAAGCCGATCAGAGAACAGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTGGGAGCACCGGCAGCATTCAAGTACTTCGACACAACAATCGACAGAAAGAGATACACAAGCACAAAGGAAGTCCTGGACGCAACACTGATCCACCAGAGCATCACAGGACTGTACGAAACAAGAATCGACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAAGCCCGAAGAAGAAGAGA AAGGTCTAG Cas9GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGTGTGTCGTT 251 transcriptGCAGGCCTTATTCGGATCCGCCACCATGGACAAGAAGTACAGCATCGGACT with 5′ UTRGGACATCGGAACAAACAGCGTCGGATGGGCAGTCATCACAGACGAATACAA of HSD, ORFGGTCCCGAGCAAGAAGTTCAAGGTCCTGGGAAACACAGACAGACACAGCAT correspondingCAAGAAGAACCTGATCGGAGCACTGCTGTTCGACAGCGGAGAAACAGCAGA to SEQ IDAGCAACAAGACTGAAGAGAACAGCAAGAAGAAGATACACAAGAAGAAAGAA NO: 250,CAGAATCTGCTACCTGCAGGAAATCTTCAGCAACGAAATGGCAAAGGTCGA KozakCGACAGCTTCTTCCACcggCTGGAAGAAAGCTTCCTGGTCGAAGAAGACAA sequence,GAAGCACGAAAGACACCCGATCTTCGGAAACATCGTCGACGAAGTCGCATA and 3′ UTRCCACGAAAAGTACCCGACAATCTACCACCTGAGAAAGAAGCTGGTCGACAG of ALBCACAGACAAGGCAGACCTGAGACTGATCTACCTGGCACTGGCACACATGATCAAGTTCAGAGGACACTTCCTGATCGAAGGAGACCTGAACCCGGACAACAGCGACGTCGACAAGCTGTTCATCCAGCTGGTCCAGACATACAACCAGCTGTTCGAAGAAAACCCGATCAACGCAAGCGGAGTCGACGCAAAGGCAATCCTGAGCGCAAGACTGAGCAAGAGCAGAAGACTGGAAAACCTGATCGCACAGCTGCCGGGAGAAAAGAAGAACGGACTGTTCGGAAACCTGATCGCACTGAGCCTGGGACTGACACCGAACTTCAAGAGCAACTTCGACCTGGCAGAAGACGCAAAGCTGCAGCTGAGCAAGGACACATACGACGACGACCTGGACAACCTGCTGGCACAGATCGGAGACCAGTACGCAGACCTGTTCCTGGCAGCAAAGAACCTGAGCGACGCAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAAATCACAAAGGCACCGCTGAGCGCAAGCATGATCAAGAGATACGACGAACACCACCAGGACCTGACACTGCTGAAGGCACTGGTCAGACAGCAGCTGCCGGAAAAGTACAAGGAAATCTTCTTCGACCAGAGCAAGAACGGATACGCAGGATACATCGACGGAGGAGCAAGCCAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAAGATGGACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAAGACCTGCTGAGAAAGCAGAGAACATTCGACAACGGAAGCATCCCGCACCAGATCCACCTGGGAGAACTGCACGCAATCCTGAGAAGACAGGAAGACTTCTACCCGTTCCTGAAGGACAACAGAGAAAAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTACGTCGGACCGCTGGCAAGAGGAAACAGCAGATTCGCATGGATGACAAGAAAGAGCGAAGAAACAATCACACCGTGGAACTTCGAAGAAGTCGTCGACAAGGGAGCAAGCGCACAGAGCTTCATCGAAAGAATGACAAACTTCGACAAGAACCTGCCGAACGAAAAGGTCCTGCCGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTACAACGAACTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCCGGCATTCCTGAGCGGAGAACAGAAGAAGGCAATCGTCGACCTGCTGTTCAAGACAAACAGAAAGGTCACAGTCAAGCAGCTGAAGGAAGACTACTTCAAGAAGATCGAATGCTTCGACAGCGTCGAAATCAGCGGAGTCGAAGACAGATTCAACGCAAGCCTGGGAACATACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGACAACGAAGAAAACGAAGACATCCTGGAAGACATCGTCCTGACACTGACACTGTTCGAAGACAGAGAAATGATCGAAGAAAGACTGAAGACATACGCACACCTGTTCGACGACAAGGTCATGAAGCAGCTGAAGAGAAGAAGATACACAGGATGGGGAAGACTGAGCAGAAAGCTGATCAACGGAATCAGAGACAAGCAGAGCGGAAAGACAATCCTGGACTTCCTGAAGAGCGACGGATTCGCAAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACATTCAAGGAAGACATCCAGAAGGCACAGGTCAGCGGACAGGGAGACAGCCTGCACGAACACATCGCAAACCTGGCAGGAAGCCCGGCAATCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAACTGGTCAAGGTCATGGGAAGACACAAGCCGGAAAACATCGTCATCGAAATGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAATGAAGAGAATCGAAGAAGGAATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACACCCGGTCGAAAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCTGCAaAACGGAAGAGACATGTACGTCGACCAGGAACTGGACATCAACAGACTGAGCGACTACGACGTCGACCACATCGTCCCGCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTCCTGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGTCCCGAGCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGACAGCTGCTGAACGCAAAGCTGATCACACAGAGAAAGTTCGACAACCTGACAAAGGCAGAGAGAGGAGGACTGAGCGAACTGGACAAGGCAGGATTCATCAAGAGACAGCTGGTCGAAACAAGACAGATCACAAAGCACGTCGCACAGATCCTGGACAGCAGAATGAACACAAAGTACGACGAAAACGACAAGCTGATCAGAGAAGTCAAGGTCATCACACTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGACTTCCAGTTCTACAAGGTCAGAGAAATCAACAACTACCACCACGCACACGACGCATACCTGAACGCAGTCGTCGGAACAGCACTGATCAAGAAGTACCCGAAGCTGGAAAGCGAATTCGTCTACGGAGACTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGAGCGAACAGGAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAAGACAGAAATCACACTGGCAAACGGAGAAATCAGAAAGAGACCGCTGATCGAAACAAACGGAGAAACAGGAGAAATCGTCTGGGACAAGGGAAGAGACTTCGCAACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCGTCAAGAAGACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAGCATCCTGCCGAAGAGAAACAGCGACAAGCTGATCGCAAGAAAGAAGGACTGGGACCCGAAGAAGTACGGAGGATTCGACAGCCCGACAGTCGCATACAGCGTCCTGGTCGTCGCAAAGGTCGAAAAGGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGCTGGGAATCACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGATCGACTTCCTGGAAGCAAAGGGATACAAGGAAGTCAAGAAGGACCTGATCATCAAGCTGCCGAAGTACAGCCTGTTCGAACTGGAAAACGGAAGAAAGAGAATGCTGGCAAGCGCAGGAGAACTGCAGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGTCAACTTCCTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCTGTTCGTCGAACAGCACAAGCACTACCTGGACGAAATCATCGAACAGATCAGCGAATTCAGCAAGAGAGTCATCCTGGCAGACGCAAACCTGGACAAGGTCCTGAGCGCATACAACAAGCACAGAGACAAGCCGATCAGAGAACAGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTGGGAGCACCGGCAGCATTCAAGTACTTCGACACAACAATCGACAGAAAGAGATACACAAGCACAAAGGAAGTCCTGGACGCAACACTGATCCACCAGAGCATCACAGGACTGTACGAAACAAGAATCGACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAAGCCCGAAGAAGAAGAGAAAGGTCTAGCTAGCCATCACATTTAAAAGCATCTCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAATAGCTTATTCATCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAACATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAATAAAAAATGGAAAGAACCTCGAG Cas9 ORFATGGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACAGCGTGGGC 252 with minimalTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAGTTCAAGGTG uridineCTGGGCAACACCGACAGACACAGCATCAAGAAGAACCTGATCGGCGCCCTG codonsCTGTTCGACAGCGGCGAGACCGCCGAGGCCACCAGACTGAAGAGAACCGCC frequentlyAGAAGAAGATACACCAGAAGAAAGAACAGAATCTGCTACCTGCAGGAGATC used inTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAG humans inGAGAGCTTCCTGGTGGAGGAGGACAAGAAGCACGAGAGACACCCCATCTTC general;GGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTAC 12.75% UCACCTGAGAAAGAAGCTGGTGGACAGCACCGACAAGGCCGACCTGAGACTG contentATCTACCTGGCCCTGGCCCACATGATCAAGTTCAGAGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAGAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGAGCGCCAGACTGAGCAAGAGCAGAAGACTGGAGAACCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAACGGCCTGTTCGGCAACCTGATCGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGACGCCAAGCTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGGCCGCCAAGAACCTGAGCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCAGCATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAGGCCCTGGTGAGACAGCAGCTGCCCGAGAAGTACAAGGAGATCTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATCGACGGCGGCGCCAGCCAGGAGGAGTTCTACAAGTTCATCAAGCCCATCCTGGAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTGAACAGAGAGGACCTGCTGAGAAAGCAGAGAACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGCGAGCTGCACGCCATCCTGAGAAGACAGGAGGACTTCTACCCCTTCCTGAAGGACAACAGAGAGAAGATCGAGAAGATCCTGACCTTCAGAATCCCCTACTACGTGGGCCCCCTGGCCAGAGGCAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAGACCATCACCCCCTGGAACTTCGAGGAGGTGGTGGACAAGGGCGCCAGCGCCCAGAGCTTCATCGAGAGAATGACCAACTTCGACAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCGAGGGCATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAGAAGGCCATCGTGGACCTGCTGTTCAAGACCAACAGAAAGGTGACCGTGAAGCAGCTGAAGGAGGACTACTTCAAGAAGATCGAGTGCTTCGACAGCGTGGAGATCAGCGGCGTGGAGGACAGATTCAACGCCAGCCTGGGCACCTACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGACATCGTGCTGACCCTGACCCTGTTCGAGGACAGAGAGATGATCGAGGAGAGACTGAAGACCTACGCCCACCTGTTCGACGACAAGGTGATGAAGCAGCTGAAGAGAAGAAGATACACCGGCTGGGGCAGACTGAGCAGAAAGCTGATCAACGGCATCAGAGACAAGCAGAGCGGCAAGACCATCCTGGACTTCCTGAAGAGCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTCAAGGAGGACATCCAGAAGGCCCAGGTGAGCGGCCAGGGCGACAGCCTGCACGAGCACATCGCCAACCTGGCCGGCAGCCCCGCCATCAAGAAGGGCATCCTGCAGACCGTGAAGGTGGTGGACGAGCTGGTGAAGGTGATGGGCAGACACAAGCCCGAGAACATCGTGATCGAGATGGCCAGAGAGAACCAGACCACCCAGAAGGGCCAGAAGAACAGCAGAGAGAGAATGAAGAGAATCGAGGAGGGCATCAAGGAGCTGGGCAGCCAGATCCTGAAGGAGCACCCCGTGGAGAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAACGGCAGAGACATGTACGTGGACCAGGAGCTGGACATCAACAGACTGAGCGACTACGACGTGGACCACATCGTGCCCCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACAGAGGCAAGAGCGACAACGTGCCCAGCGAGGAGGTGGTGAAGAAGATGAAGAACTACTGGAGACAGCTGCTGAACGCCAAGCTGATCACCCAGAGAAAGTTCGACAACCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAGCTGGACAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAGACCAGACAGATCACCAAGCACGTGGCCCAGATCCTGGACAGCAGAATGAACACCAAGTACGACGAGAACGACAAGCTGATCAGAGAGGTGAAGGTGATCACCCTGAAGAGCAAGCTGGTGAGCGACTTCAGAAAGGACTTCCAGTTCTACAAGGTGAGAGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTGGTGGGCACCGCCCTGATCAAGAAGTACCCCAAGCTGGAGAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGAGAAAGATGATCGCCAAGAGCGAGCAGGAGATCGGCAAGGCCACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAAGACCGAGATCACCCTGGCCAACGGCGAGATCAGAAAGAGACCCCTGATCGAGACCAACGGCGAGACCGGCGAGATCGTGTGGGACAAGGGCAGAGACTTCGCCACCGTGAGAAAGGTGCTGAGCATGCCCCAGGTGAACATCGTGAAGAAGACCGAGGTGCAGACCGGCGGCTTCAGCAAGGAGAGCATCCTGCCCAAGAGAAACAGCGACAAGCTGATCGCCAGAAAGAAGGACTGGGACCCCAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTACAGCGTGCTGGTGGTGGCCAAGGTGGAGAAGGGCAAGAGCAAGAAGCTGAAGAGCGTGAAGGAGCTGCTGGGCATCACCATCATGGAGAGAAGCAGCTTCGAGAAGAACCCCATCGACTTCCTGGAGGCCAAGGGCTACAAGGAGGTGAAGAAGGACCTGATCATCAAGCTGCCCAAGTACAGCCTGTTCGAGCTGGAGAACGGCAGAAAGAGAATGCTGGCCAGCGCCGGCGAGCTGCAGAAGGGCAACGAGCTGGCCCTGCCCAGCAAGTACGTGAACTTCCTGTACCTGGCCAGCCACTACGAGAAGCTGAAGGGCAGCCCCGAGGACAACGAGCAGAAGCAGCTGTTCGTGGAGCAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCAGCAAGAGAGTGATCCTGGCCGACGCCAACCTGGACAAGGTGCTGAGCGCCTACAACAAGCACAGAGACAAGCCCATCAGAGAGCAGGCCGAGAACATCATCCACCTGTTCACCCTGACCAACCTGGGCGCCCCCGCCGCCTTCAAGTACTTCGACACCACCATCGACAGAAAGAGATACACCAGCACCAAGGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACCAGAATCGACCTGAGCCAGCTGGGCGGCGACGGCGGCGGCAGCCCCAAGAAGAAGAGA AAGGTGTGA Cas9GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGTGTGTCGTT 253 transcriptGCAGGCCTTATTCGGATCCGCCACCATGGACAAGAAGTACAGCATCGGCCT with 5′ UTRGGACATCGGCACCAACAGCGTGGGCTGGGCCGTGATCACCGACGAGTACAA of HSD, ORFGGTGCCCAGCAAGAAGTTCAAGGTGCTGGGCAACACCGACAGACACAGCAT correspondingCAAGAAGAACCTGATCGGCGCCCTGCTGTTCGACAGCGGCGAGACCGCCGA to SEQ IDGGCCACCAGACTGAAGAGAACCGCCAGAAGAAGATACACCAGAAGAAAGAA NO: 252,CAGAATCTGCTACCTGCAGGAGATCTTCAGCAACGAGATGGCCAAGGTGGA KozakCGACAGCTTCTTCCACAGACTGGAGGAGAGCTTCCTGGTGGAGGAGGACAA sequence,GAAGCACGAGAGACACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTA and 3′ UTRCCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAGCTGGTGGACAG of ALBCACCGACAAGGCCGACCTGAGACTGATCTACCTGGCCCTGGCCCACATGATCAAGTTCAGAGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAGAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGAGCGCCAGACTGAGCAAGAGCAGAAGACTGGAGAACCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAACGGCCTGTTCGGCAACCTGATCGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGACGCCAAGCTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGGCCGCCAAGAACCTGAGCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCAGCATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAGGCCCTGGTGAGACAGCAGCTGCCCGAGAAGTACAAGGAGATCTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATCGACGGCGGCGCCAGCCAGGAGGAGTTCTACAAGTTCATCAAGCCCATCCTGGAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTGAACAGAGAGGACCTGCTGAGAAAGCAGAGAACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGCGAGCTGCACGCCATCCTGAGAAGACAGGAGGACTTCTACCCCTTCCTGAAGGACAACAGAGAGAAGATCGAGAAGATCCTGACCTTCAGAATCCCCTACTACGTGGGCCCCCTGGCCAGAGGCAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAGACCATCACCCCCTGGAACTTCGAGGAGGTGGTGGACAAGGGCGCCAGCGCCCAGAGCTTCATCGAGAGAATGACCAACTTCGACAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCGAGGGCATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAGAAGGCCATCGTGGACCTGCTGTTCAAGACCAACAGAAAGGTGACCGTGAAGCAGCTGAAGGAGGACTACTTCAAGAAGATCGAGTGCTTCGACAGCGTGGAGATCAGCGGCGTGGAGGACAGATTCAACGCCAGCCTGGGCACCTACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGACATCGTGCTGACCCTGACCCTGTTCGAGGACAGAGAGATGATCGAGGAGAGACTGAAGACCTACGCCCACCTGTTCGACGACAAGGTGATGAAGCAGCTGAAGAGAAGAAGATACACCGGCTGGGGCAGACTGAGCAGAAAGCTGATCAACGGCATCAGAGACAAGCAGAGCGGCAAGACCATCCTGGACTTCCTGAAGAGCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTCAAGGAGGACATCCAGAAGGCCCAGGTGAGCGGCCAGGGCGACAGCCTGCACGAGCACATCGCCAACCTGGCCGGCAGCCCCGCCATCAAGAAGGGCATCCTGCAGACCGTGAAGGTGGTGGACGAGCTGGTGAAGGTGATGGGCAGACACAAGCCCGAGAACATCGTGATCGAGATGGCCAGAGAGAACCAGACCACCCAGAAGGGCCAGAAGAACAGCAGAGAGAGAATGAAGAGAATCGAGGAGGGCATCAAGGAGCTGGGCAGCCAGATCCTGAAGGAGCACCCCGTGGAGAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAACGGCAGAGACATGTACGTGGACCAGGAGCTGGACATCAACAGACTGAGCGACTACGACGTGGACCACATCGTGCCCCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACAGAGGCAAGAGCGACAACGTGCCCAGCGAGGAGGTGGTGAAGAAGATGAAGAACTACTGGAGACAGCTGCTGAACGCCAAGCTGATCACCCAGAGAAAGTTCGACAACCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAGCTGGACAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAGACCAGACAGATCACCAAGCACGTGGCCCAGATCCTGGACAGCAGAATGAACACCAAGTACGACGAGAACGACAAGCTGATCAGAGAGGTGAAGGTGATCACCCTGAAGAGCAAGCTGGTGAGCGACTTCAGAAAGGACTTCCAGTTCTACAAGGTGAGAGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTGGTGGGCACCGCCCTGATCAAGAAGTACCCCAAGCTGGAGAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGAGAAAGATGATCGCCAAGAGCGAGCAGGAGATCGGCAAGGCCACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAAGACCGAGATCACCCTGGCCAACGGCGAGATCAGAAAGAGACCCCTGATCGAGACCAACGGCGAGACCGGCGAGATCGTGTGGGACAAGGGCAGAGACTTCGCCACCGTGAGAAAGGTGCTGAGCATGCCCCAGGTGAACATCGTGAAGAAGACCGAGGTGCAGACCGGCGGCTTCAGCAAGGAGAGCATCCTGCCCAAGAGAAACAGCGACAAGCTGATCGCCAGAAAGAAGGACTGGGACCCCAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTACAGCGTGCTGGTGGTGGCCAAGGTGGAGAAGGGCAAGAGCAAGAAGCTGAAGAGCGTGAAGGAGCTGCTGGGCATCACCATCATGGAGAGAAGCAGCTTCGAGAAGAACCCCATCGACTTCCTGGAGGCCAAGGGCTACAAGGAGGTGAAGAAGGACCTGATCATCAAGCTGCCCAAGTACAGCCTGTTCGAGCTGGAGAACGGCAGAAAGAGAATGCTGGCCAGCGCCGGCGAGCTGCAGAAGGGCAACGAGCTGGCCCTGCCCAGCAAGTACGTGAACTTCCTGTACCTGGCCAGCCACTACGAGAAGCTGAAGGGCAGCCCCGAGGACAACGAGCAGAAGCAGCTGTTCGTGGAGCAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCAGCAAGAGAGTGATCCTGGCCGACGCCAACCTGGACAAGGTGCTGAGCGCCTACAACAAGCACAGAGACAAGCCCATCAGAGAGCAGGCCGAGAACATCATCCACCTGTTCACCCTGACCAACCTGGGCGCCCCCGCCGCCTTCAAGTACTTCGACACCACCATCGACAGAAAGAGATACACCAGCACCAAGGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACCAGAATCGACCTGAGCCAGCTGGGCGGCGACGGCGGCGGCAGCCCCAAGAAGAAGAGAAAGGTGTGACTAGCCATCACATTTAAAAGCATCTCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAATAGCTTATTCATCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAACATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAATAAAAAATGGAAAGAACCTCGAG Cas9 ORFATGGACAAAAAATACAGCATAGGGCTAGACATAGGGACGAACAGCGTAGGG 254 with minimalTGGGCGGTAATAACGGACGAATACAAAGTACCGAGCAAAAAATTCAAAGTA uridineCTAGGGAACACGGACCGACACAGCATAAAAAAAAACCTAATAGGGGCGCTA codonsCTATTCGACAGCGGGGAAACGGCGGAAGCGACGCGACTAAAACGAACGGCG infrequentlyCGACGACGATACACGCGACGAAAAAACCGAATATGCTACCTACAAGAAATA used inTTCAGCAACGAAATGGCGAAAGTAGACGACAGCTTCTTCCACCGACTAGAA humans inGAAAGCTTCCTAGTAGAAGAAGACAAAAAACACGAACGACACCCGATATTC general;GGGAACATAGTAGACGAAGTAGCGTACCACGAAAAATACCCGACGATATAC 12.75% UCACCTACGAAAAAAACTAGTAGACAGCACGGACAAAGCGGACCTACGACTA contentATATACCTAGCGCTAGCGCACATGATAAAATTCCGAGGGCACTTCCTAATAGAAGGGGACCTAAACCCGGACAACAGCGACGTAGACAAACTATTCATACAACTAGTACAAACGTACAACCAACTATTCGAAGAAAACCCGATAAACGCGAGCGGGGTAGACGCGAAAGCGATACTAAGCGCGCGACTAAGCAAAAGCCGACGACTAGAAAACCTAATAGCGCAACTACCGGGGGAAAAAAAAAACGGGCTATTCGGGAACCTAATAGCGCTAAGCCTAGGGCTAACGCCGAACTTCAAAAGCAACTTCGACCTAGCGGAAGACGCGAAACTACAACTAAGCAAAGACACGTACGACGACGACCTAGACAACCTACTAGCGCAAATAGGGGACCAATACGCGGACCTATTCCTAGCGGCGAAAAACCTAAGCGACGCGATACTACTAAGCGACATACTACGAGTAAACACGGAAATAACGAAAGCGCCGCTAAGCGCGAGCATGATAAAACGATACGACGAACACCACCAAGACCTAACGCTACTAAAAGCGCTAGTACGACAACAACTACCGGAAAAATACAAAGAAATATTCTTCGACCAAAGCAAAAACGGGTACGCGGGGTACATAGACGGGGGGGCGAGCCAAGAAGAATTCTACAAATTCATAAAACCGATACTAGAAAAAATGGACGGGACGGAAGAACTACTAGTAAAACTAAACCGAGAAGACCTACTACGAAAACAACGAACGTTCGACAACGGGAGCATACCGCACCAAATACACCTAGGGGAACTACACGCGATACTACGACGACAAGAAGACTTCTACCCGTTCCTAAAAGACAACCGAGAAAAAATAGAAAAAATACTAACGTTCCGAATACCGTACTACGTAGGGCCGCTAGCGCGAGGGAACAGCCGATTCGCGTGGATGACGCGAAAAAGCGAAGAAACGATAACGCCGTGGAACTTCGAAGAAGTAGTAGACAAAGGGGCGAGCGCGCAAAGCTTCATAGAACGAATGACGAACTTCGACAAAAACCTACCGAACGAAAAAGTACTACCGAAACACAGCCTACTATACGAATACTTCACGGTATACAACGAACTAACGAAAGTAAAATACGTAACGGAAGGGATGCGAAAACCGGCGTTCCTAAGCGGGGAACAAAAAAAAGCGATAGTAGACCTACTATTCAAAACGAACCGAAAAGTAACGGTAAAACAACTAAAAGAAGACTACTTCAAAAAAATAGAATGCTTCGACAGCGTAGAAATAAGCGGGGTAGAAGACCGATTCAACGCGAGCCTAGGGACGTACCACGACCTACTAAAAATAATAAAAGACAAAGACTTCCTAGACAACGAAGAAAACGAAGACATACTAGAAGACATAGTACTAACGCTAACGCTATTCGAAGACCGAGAAATGATAGAAGAACGACTAAAAACGTACGCGCACCTATTCGACGACAAAGTAATGAAACAACTAAAACGACGACGATACACGGGGTGGGGGCGACTAAGCCGAAAACTAATAAACGGGATACGAGACAAACAAAGCGGGAAAACGATACTAGACTTCCTAAAAAGCGACGGGTTCGCGAACCGAAACTTCATGCAACTAATACACGACGACAGCCTAACGTTCAAAGAAGACATACAAAAAGCGCAAGTAAGCGGGCAAGGGGACAGCCTACACGAACACATAGCGAACCTAGCGGGGAGCCCGGCGATAAAAAAAGGGATACTACAAACGGTAAAAGTAGTAGACGAACTAGTAAAAGTAATGGGGCGACACAAACCGGAAAACATAGTAATAGAAATGGCGCGAGAAAACCAAACGACGCAAAAAGGGCAAAAAAACAGCCGAGAACGAATGAAACGAATAGAAGAAGGGATAAAAGAACTAGGGAGCCAAATACTAAAAGAACACCCGGTAGAAAACACGCAACTACAAAACGAAAAACTATACCTATACTACCTACAAAACGGGCGAGACATGTACGTAGACCAAGAACTAGACATAAACCGACTAAGCGACTACGACGTAGACCACATAGTACCGCAAAGCTTCCTAAAAGACGACAGCATAGACAACAAAGTACTAACGCGAAGCGACAAAAACCGAGGGAAAAGCGACAACGTACCGAGCGAAGAAGTAGTAAAAAAAATGAAAAACTACTGGCGACAACTACTAAACGCGAAACTAATAACGCAACGAAAATTCGACAACCTAACGAAAGCGGAACGAGGGGGGCTAAGCGAACTAGACAAAGCGGGGTTCATAAAACGACAACTAGTAGAAACGCGACAAATAACGAAACACGTAGCGCAAATACTAGACAGCCGAATGAACACGAAATACGACGAAAACGACAAACTAATACGAGAAGTAAAAGTAATAACGCTAAAAAGCAAACTAGTAAGCGACTTCCGAAAAGACTTCCAATTCTACAAAGTACGAGAAATAAACAACTACCACCACGCGCACGACGCGTACCTAAACGCGGTAGTAGGGACGGCGCTAATAAAAAAATACCCGAAACTAGAAAGCGAATTCGTATACGGGGACTACAAAGTATACGACGTACGAAAAATGATAGCGAAAAGCGAACAAGAAATAGGGAAAGCGACGGCGAAATACTTCTTCTACAGCAACATAATGAACTTCTTCAAAACGGAAATAACGCTAGCGAACGGGGAAATACGAAAACGACCGCTAATAGAAACGAACGGGGAAACGGGGGAAATAGTATGGGACAAAGGGCGAGACTTCGCGACGGTACGAAAAGTACTAAGCATGCCGCAAGTAAACATAGTAAAAAAAACGGAAGTACAAACGGGGGGGTTCAGCAAAGAAAGCATACTACCGAAACGAAACAGCGACAAACTAATAGCGCGAAAAAAAGACTGGGACCCGAAAAAATACGGGGGGTTCGACAGCCCGACGGTAGCGTACAGCGTACTAGTAGTAGCGAAAGTAGAAAAAGGGAAAAGCAAAAAACTAAAAAGCGTAAAAGAACTACTAGGGATAACGATAATGGAACGAAGCAGCTTCGAAAAAAACCCGATAGACTTCCTAGAAGCGAAAGGGTACAAAGAAGTAAAAAAAGACCTAATAATAAAACTACCGAAATACAGCCTATTCGAACTAGAAAACGGGCGAAAACGAATGCTAGCGAGCGCGGGGGAACTACAAAAAGGGAACGAACTAGCGCTACCGAGCAAATACGTAAACTTCCTATACCTAGCGAGCCACTACGAAAAACTAAAAGGGAGCCCGGAAGACAACGAACAAAAACAACTATTCGTAGAACAACACAAACACTACCTAGACGAAATAATAGAACAAATAAGCGAATTCAGCAAACGAGTAATACTAGCGGACGCGAACCTAGACAAAGTACTAAGCGCGTACAACAAACACCGAGACAAACCGATACGAGAACAAGCGGAAAACATAATACACCTATTCACGCTAACGAACCTAGGGGCGCCGGCGGCGTTCAAATACTTCGACACGACGATAGACCGAAAACGATACACGAGCACGAAAGAAGTACTAGACGCGACGCTAATACACCAAAGCATAACGGGGCTATACGAAACGCGAATAGACCTAAGCCAACTAGGGGGGGACGGGGGGGGGAGCCCGAAAAAAAAACGA AAAGTATGA Cas9GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGTGTGTCGTT 255 transcriptGCAGGCCTTATTCGGATCCGCCACCATGGACAAAAAATACAGCATAGGGCT with 5′ UTRAGACATAGGGACGAACAGCGTAGGGTGGGCGGTAATAACGGACGAATACAA of HSD, ORFAGTACCGAGCAAAAAATTCAAAGTACTAGGGAACACGGACCGACACAGCAT correspondingAAAAAAAAACCTAATAGGGGCGCTACTATTCGACAGCGGGGAAACGGCGGA to SEQ IDAGCGACGCGACTAAAACGAACGGCGCGACGACGATACACGCGACGAAAAAA NO: 254,CCGAATATGCTACCTACAAGAAATATTCAGCAACGAAATGGCGAAAGTAGA KozakCGACAGCTTCTTCCACCGACTAGAAGAAAGCTTCCTAGTAGAAGAAGACAA sequence,AAAACACGAACGACACCCGATATTCGGGAACATAGTAGACGAAGTAGCGTA and 3′ UTRCCACGAAAAATACCCGACGATATACCACCTACGAAAAAAACTAGTAGACAG of ALBCACGGACAAAGCGGACCTACGACTAATATACCTAGCGCTAGCGCACATGATAAAATTCCGAGGGCACTTCCTAATAGAAGGGGACCTAAACCCGGACAACAGCGACGTAGACAAACTATTCATACAACTAGTACAAACGTACAACCAACTATTCGAAGAAAACCCGATAAACGCGAGCGGGGTAGACGCGAAAGCGATACTAAGCGCGCGACTAAGCAAAAGCCGACGACTAGAAAACCTAATAGCGCAACTACCGGGGGAAAAAAAAAACGGGCTATTCGGGAACCTAATAGCGCTAAGCCTAGGGCTAACGCCGAACTTCAAAAGCAACTTCGACCTAGCGGAAGACGCGAAACTACAACTAAGCAAAGACACGTACGACGACGACCTAGACAACCTACTAGCGCAAATAGGGGACCAATACGCGGACCTATTCCTAGCGGCGAAAAACCTAAGCGACGCGATACTACTAAGCGACATACTACGAGTAAACACGGAAATAACGAAAGCGCCGCTAAGCGCGAGCATGATAAAACGATACGACGAACACCACCAAGACCTAACGCTACTAAAAGCGCTAGTACGACAACAACTACCGGAAAAATACAAAGAAATATTCTTCGACCAAAGCAAAAACGGGTACGCGGGGTACATAGACGGGGGGGCGAGCCAAGAAGAATTCTACAAATTCATAAAACCGATACTAGAAAAAATGGACGGGACGGAAGAACTACTAGTAAAACTAAACCGAGAAGACCTACTACGAAAACAACGAACGTTCGACAACGGGAGCATACCGCACCAAATACACCTAGGGGAACTACACGCGATACTACGACGACAAGAAGACTTCTACCCGTTCCTAAAAGACAACCGAGAAAAAATAGAAAAAATACTAACGTTCCGAATACCGTACTACGTAGGGCCGCTAGCGCGAGGGAACAGCCGATTCGCGTGGATGACGCGAAAAAGCGAAGAAACGATAACGCCGTGGAACTTCGAAGAAGTAGTAGACAAAGGGGCGAGCGCGCAAAGCTTCATAGAACGAATGACGAACTTCGACAAAAACCTACCGAACGAAAAAGTACTACCGAAACACAGCCTACTATACGAATACTTCACGGTATACAACGAACTAACGAAAGTAAAATACGTAACGGAAGGGATGCGAAAACCGGCGTTCCTAAGCGGGGAACAAAAAAAAGCGATAGTAGACCTACTATTCAAAACGAACCGAAAAGTAACGGTAAAACAACTAAAAGAAGACTACTTCAAAAAAATAGAATGCTTCGACAGCGTAGAAATAAGCGGGGTAGAAGACCGATTCAACGCGAGCCTAGGGACGTACCACGACCTACTAAAAATAATAAAAGACAAAGACTTCCTAGACAACGAAGAAAACGAAGACATACTAGAAGACATAGTACTAACGCTAACGCTATTCGAAGACCGAGAAATGATAGAAGAACGACTAAAAACGTACGCGCACCTATTCGACGACAAAGTAATGAAACAACTAAAACGACGACGATACACGGGGTGGGGGCGACTAAGCCGAAAACTAATAAACGGGATACGAGACAAACAAAGCGGGAAAACGATACTAGACTTCCTAAAAAGCGACGGGTTCGCGAACCGAAACTTCATGCAACTAATACACGACGACAGCCTAACGTTCAAAGAAGACATACAAAAAGCGCAAGTAAGCGGGCAAGGGGACAGCCTACACGAACACATAGCGAACCTAGCGGGGAGCCCGGCGATAAAAAAAGGGATACTACAAACGGTAAAAGTAGTAGACGAACTAGTAAAAGTAATGGGGCGACACAAACCGGAAAACATAGTAATAGAAATGGCGCGAGAAAACCAAACGACGCAAAAAGGGCAAAAAAACAGCCGAGAACGAATGAAACGAATAGAAGAAGGGATAAAAGAACTAGGGAGCCAAATACTAAAAGAACACCCGGTAGAAAACACGCAACTACAAAACGAAAAACTATACCTATACTACCTACAAAACGGGCGAGACATGTACGTAGACCAAGAACTAGACATAAACCGACTAAGCGACTACGACGTAGACCACATAGTACCGCAAAGCTTCCTAAAAGACGACAGCATAGACAACAAAGTACTAACGCGAAGCGACAAAAACCGAGGGAAAAGCGACAACGTACCGAGCGAAGAAGTAGTAAAAAAAATGAAAAACTACTGGCGACAACTACTAAACGCGAAACTAATAACGCAACGAAAATTCGACAACCTAACGAAAGCGGAACGAGGGGGGCTAAGCGAACTAGACAAAGCGGGGTTCATAAAACGACAACTAGTAGAAACGCGACAAATAACGAAACACGTAGCGCAAATACTAGACAGCCGAATGAACACGAAATACGACGAAAACGACAAACTAATACGAGAAGTAAAAGTAATAACGCTAAAAAGCAAACTAGTAAGCGACTTCCGAAAAGACTTCCAATTCTACAAAGTACGAGAAATAAACAACTACCACCACGCGCACGACGCGTACCTAAACGCGGTAGTAGGGACGGCGCTAATAAAAAAATACCCGAAACTAGAAAGCGAATTCGTATACGGGGACTACAAAGTATACGACGTACGAAAAATGATAGCGAAAAGCGAACAAGAAATAGGGAAAGCGACGGCGAAATACTTCTTCTACAGCAACATAATGAACTTCTTCAAAACGGAAATAACGCTAGCGAACGGGGAAATACGAAAACGACCGCTAATAGAAACGAACGGGGAAACGGGGGAAATAGTATGGGACAAAGGGCGAGACTTCGCGACGGTACGAAAAGTACTAAGCATGCCGCAAGTAAACATAGTAAAAAAAACGGAAGTACAAACGGGGGGGTTCAGCAAAGAAAGCATACTACCGAAACGAAACAGCGACAAACTAATAGCGCGAAAAAAAGACTGGGACCCGAAAAAATACGGGGGGTTCGACAGCCCGACGGTAGCGTACAGCGTACTAGTAGTAGCGAAAGTAGAAAAAGGGAAAAGCAAAAAACTAAAAAGCGTAAAAGAACTACTAGGGATAACGATAATGGAACGAAGCAGCTTCGAAAAAAACCCGATAGACTTCCTAGAAGCGAAAGGGTACAAAGAAGTAAAAAAAGACCTAATAATAAAACTACCGAAATACAGCCTATTCGAACTAGAAAACGGGCGAAAACGAATGCTAGCGAGCGCGGGGGAACTACAAAAAGGGAACGAACTAGCGCTACCGAGCAAATACGTAAACTTCCTATACCTAGCGAGCCACTACGAAAAACTAAAAGGGAGCCCGGAAGACAACGAACAAAAACAACTATTCGTAGAACAACACAAACACTACCTAGACGAAATAATAGAACAAATAAGCGAATTCAGCAAACGAGTAATACTAGCGGACGCGAACCTAGACAAAGTACTAAGCGCGTACAACAAACACCGAGACAAACCGATACGAGAACAAGCGGAAAACATAATACACCTATTCACGCTAACGAACCTAGGGGCGCCGGCGGCGTTCAAATACTTCGACACGACGATAGACCGAAAACGATACACGAGCACGAAAGAAGTACTAGACGCGACGCTAATACACCAAAGCATAACGGGGCTATACGAAACGCGAATAGACCTAAGCCAACTAGGGGGGGACGGGGGGGGGAGCCCGAAAAAAAAACGAAAAGTATGACTAGCCATCACATTTAAAAGCATCTCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAATAGCTTATTCATCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAACATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAATAAAAAATGGAAAGAACCTCGAG Cas9AGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGTGTGTCGTT 256 transcriptGCAGGCCTTATTCGGATCCGCCACCATGGACAAGAAGTACAGCATCGGACT with AGG asGGACATCGGAACAAACAGCGTCGGATGGGCAGTCATCACAGACGAATACAA first threeGGTCCCGAGCAAGAAGTTCAAGGTCCTGGGAAACACAGACAGACACAGCAT nucleotidesCAAGAAGAACCTGATCGGAGCACTGCTGTTCGACAGCGGAGAAACAGCAGA for use withAGCAACAAGACTGAAGAGAACAGCAAGAAGAAGATACACAAGAAGAAAGAA CleanCap ™,CAGAATCTGCTACCTGCAGGAAATCTTCAGCAACGAAATGGCAAAGGTCGA 5′ UTR ofCGACAGCTTCTTCCACAGACTGGAAGAAAGCTTCCTGGTCGAAGAAGACAA HSD, ORFGAAGCACGAAAGACACCCGATCTTCGGAAACATCGTCGACGAAGTCGCATA correspondingCCACGAAAAGTACCCGACAATCTACCACCTGAGAAAGAAGCTGGTCGACAG to SEQ IDCACAGACAAGGCAGACCTGAGACTGATCTACCTGGCACTGGCACACATGAT NO: 204,CAAGTTCAGAGGACACTTCCTGATCGAAGGAGACCTGAACCCGGACAACAG KozakCGACGTCGACAAGCTGTTCATCCAGCTGGTCCAGACATACAACCAGCTGTT sequence,CGAAGAAAACCCGATCAACGCAAGCGGAGTCGACGCAAAGGCAATCCTGAG and 3′ UTRCGCAAGACTGAGCAAGAGCAGAAGACTGGAAAACCTGATCGCACAGCTGCC of ALBGGGAGAAAAGAAGAACGGACTGTTCGGAAACCTGATCGCACTGAGCCTGGGACTGACACCGAACTTCAAGAGCAACTTCGACCTGGCAGAAGACGCAAAGCTGCAGCTGAGCAAGGACACATACGACGACGACCTGGACAACCTGCTGGCACAGATCGGAGACCAGTACGCAGACCTGTTCCTGGCAGCAAAGAACCTGAGCGACGCAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAAATCACAAAGGCACCGCTGAGCGCAAGCATGATCAAGAGATACGACGAACACCACCAGGACCTGACACTGCTGAAGGCACTGGTCAGACAGCAGCTGCCGGAAAAGTACAAGGAAATCTTCTTCGACCAGAGCAAGAACGGATACGCAGGATACATCGACGGAGGAGCAAGCCAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAAGATGGACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAAGACCTGCTGAGAAAGCAGAGAACATTCGACAACGGAAGCATCCCGCACCAGATCCACCTGGGAGAACTGCACGCAATCCTGAGAAGACAGGAAGACTTCTACCCGTTCCTGAAGGACAACAGAGAAAAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTACGTCGGACCGCTGGCAAGAGGAAACAGCAGATTCGCATGGATGACAAGAAAGAGCGAAGAAACAATCACACCGTGGAACTTCGAAGAAGTCGTCGACAAGGGAGCAAGCGCACAGAGCTTCATCGAAAGAATGACAAACTTCGACAAGAACCTGCCGAACGAAAAGGTCCTGCCGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTACAACGAACTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCCGGCATTCCTGAGCGGAGAACAGAAGAAGGCAATCGTCGACCTGCTGTTCAAGACAAACAGAAAGGTCACAGTCAAGCAGCTGAAGGAAGACTACTTCAAGAAGATCGAATGCTTCGACAGCGTCGAAATCAGCGGAGTCGAAGACAGATTCAACGCAAGCCTGGGAACATACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGACAACGAAGAAAACGAAGACATCCTGGAAGACATCGTCCTGACACTGACACTGTTCGAAGACAGAGAAATGATCGAAGAAAGACTGAAGACATACGCACACCTGTTCGACGACAAGGTCATGAAGCAGCTGAAGAGAAGAAGATACACAGGATGGGGAAGACTGAGCAGAAAGCTGATCAACGGAATCAGAGACAAGCAGAGCGGAAAGACAATCCTGGACTTCCTGAAGAGCGACGGATTCGCAAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACATTCAAGGAAGACATCCAGAAGGCACAGGTCAGCGGACAGGGAGACAGCCTGCACGAACACATCGCAAACCTGGCAGGAAGCCCGGCAATCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAACTGGTCAAGGTCATGGGAAGACACAAGCCGGAAAACATCGTCATCGAAATGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAATGAAGAGAATCGAAGAAGGAATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACACCCGGTCGAAAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCTGCAGAACGGAAGAGACATGTACGTCGACCAGGAACTGGACATCAACAGACTGAGCGACTACGACGTCGACCACATCGTCCCGCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTCCTGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGTCCCGAGCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGACAGCTGCTGAACGCAAAGCTGATCACACAGAGAAAGTTCGACAACCTGACAAAGGCAGAGAGAGGAGGACTGAGCGAACTGGACAAGGCAGGATTCATCAAGAGACAGCTGGTCGAAACAAGACAGATCACAAAGCACGTCGCACAGATCCTGGACAGCAGAATGAACACAAAGTACGACGAAAACGACAAGCTGATCAGAGAAGTCAAGGTCATCACACTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGACTTCCAGTTCTACAAGGTCAGAGAAATCAACAACTACCACCACGCACACGACGCATACCTGAACGCAGTCGTCGGAACAGCACTGATCAAGAAGTACCCGAAGCTGGAAAGCGAATTCGTCTACGGAGACTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGAGCGAACAGGAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAAGACAGAAATCACACTGGCAAACGGAGAAATCAGAAAGAGACCGCTGATCGAAACAAACGGAGAAACAGGAGAAATCGTCTGGGACAAGGGAAGAGACTTCGCAACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCGTCAAGAAGACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAGCATCCTGCCGAAGAGAAACAGCGACAAGCTGATCGCAAGAAAGAAGGACTGGGACCCGAAGAAGTACGGAGGATTCGACAGCCCGACAGTCGCATACAGCGTCCTGGTCGTCGCAAAGGTCGAAAAGGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGCTGGGAATCACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGATCGACTTCCTGGAAGCAAAGGGATACAAGGAAGTCAAGAAGGACCTGATCATCAAGCTGCCGAAGTACAGCCTGTTCGAACTGGAAAACGGAAGAAAGAGAATGCTGGCAAGCGCAGGAGAACTGCAGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGTCAACTTCCTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCTGTTCGTCGAACAGCACAAGCACTACCTGGACGAAATCATCGAACAGATCAGCGAATTCAGCAAGAGAGTCATCCTGGCAGACGCAAACCTGGACAAGGTCCTGAGCGCATACAACAAGCACAGAGACAAGCCGATCAGAGAACAGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTGGGAGCACCGGCAGCATTCAAGTACTTCGACACAACAATCGACAGAAAGAGATACACAAGCACAAAGGAAGTCCTGGACGCAACACTGATCCACCAGAGCATCACAGGACTGTACGAAACAAGAATCGACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAAGCCCGAAGAAGAAGAGAAAGGTCTAGCTAGCCATCACATTTAAAAGCATCTCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAATAGCTTATTCATCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAACATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAATAAAAAATGGAAAGAACCTCGAG Cas9GGGCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGA 257 transcriptCACCGGGACCGATCCAGCCTCCGCGGCCGGGAACGGTGCATTGGAACGCGG with 5′ UTRATTCCCCGTGCCAAGAGTGACTCACCGTCCTTGACACGGCCACCATGGACA from CMV,AGAAGTACAGCATCGGACTGGACATCGGAACAAACAGCGTCGGATGGGCAG ORFTCATCACAGACGAATACAAGGTCCCGAGCAAGAAGTTCAAGGTCCTGGGAA correspondingACACAGACAGACACAGCATCAAGAAGAACCTGATCGGAGCACTGCTGTTCG to SEQ IDACAGCGGAGAAACAGCAGAAGCAACAAGACTGAAGAGAACAGCAAGAAGAA NO: 204,GATACACAAGAAGAAAGAACAGAATCTGCTACCTGCAGGAAATCTTCAGCA KozakACGAAATGGCAAAGGTCGACGACAGCTTCTTCCACAGACTGGAAGAAAGCT sequence,TCCTGGTCGAAGAAGACAAGAAGCACGAAAGACACCCGATCTTCGGAAACA and 3′ UTRTCGTCGACGAAGTCGCATACCACGAAAAGTACCCGACAATCTACCACCTGA of ALBGAAAGAAGCTGGTCGACAGCACAGACAAGGCAGACCTGAGACTGATCTACCTGGCACTGGCACACATGATCAAGTTCAGAGGACACTTCCTGATCGAAGGAGACCTGAACCCGGACAACAGCGACGTCGACAAGCTGTTCATCCAGCTGGTCCAGACATACAACCAGCTGTTCGAAGAAAACCCGATCAACGCAAGCGGAGTCGACGCAAAGGCAATCCTGAGCGCAAGACTGAGCAAGAGCAGAAGACTGGAAAACCTGATCGCACAGCTGCCGGGAGAAAAGAAGAACGGACTGTTCGGAAACCTGATCGCACTGAGCCTGGGACTGACACCGAACTTCAAGAGCAACTTCGACCTGGCAGAAGACGCAAAGCTGCAGCTGAGCAAGGACACATACGACGACGACCTGGACAACCTGCTGGCACAGATCGGAGACCAGTACGCAGACCTGTTCCTGGCAGCAAAGAACCTGAGCGACGCAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAAATCACAAAGGCACCGCTGAGCGCAAGCATGATCAAGAGATACGACGAACACCACCAGGACCTGACACTGCTGAAGGCACTGGTCAGACAGCAGCTGCCGGAAAAGTACAAGGAAATCTTCTTCGACCAGAGCAAGAACGGATACGCAGGATACATCGACGGAGGAGCAAGCCAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAAGATGGACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAAGACCTGCTGAGAAAGCAGAGAACATTCGACAACGGAAGCATCCCGCACCAGATCCACCTGGGAGAACTGCACGCAATCCTGAGAAGACAGGAAGACTTCTACCCGTTCCTGAAGGACAACAGAGAAAAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTACGTCGGACCGCTGGCAAGAGGAAACAGCAGATTCGCATGGATGACAAGAAAGAGCGAAGAAACAATCACACCGTGGAACTTCGAAGAAGTCGTCGACAAGGGAGCAAGCGCACAGAGCTTCATCGAAAGAATGACAAACTTCGACAAGAACCTGCCGAACGAAAAGGTCCTGCCGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTACAACGAACTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCCGGCATTCCTGAGCGGAGAACAGAAGAAGGCAATCGTCGACCTGCTGTTCAAGACAAACAGAAAGGTCACAGTCAAGCAGCTGAAGGAAGACTACTTCAAGAAGATCGAATGCTTCGACAGCGTCGAAATCAGCGGAGTCGAAGACAGATTCAACGCAAGCCTGGGAACATACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGACAACGAAGAAAACGAAGACATCCTGGAAGACATCGTCCTGACACTGACACTGTTCGAAGACAGAGAAATGATCGAAGAAAGACTGAAGACATACGCACACCTGTTCGACGACAAGGTCATGAAGCAGCTGAAGAGAAGAAGATACACAGGATGGGGAAGACTGAGCAGAAAGCTGATCAACGGAATCAGAGACAAGCAGAGCGGAAAGACAATCCTGGACTTCCTGAAGAGCGACGGATTCGCAAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACATTCAAGGAAGACATCCAGAAGGCACAGGTCAGCGGACAGGGAGACAGCCTGCACGAACACATCGCAAACCTGGCAGGAAGCCCGGCAATCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAACTGGTCAAGGTCATGGGAAGACACAAGCCGGAAAACATCGTCATCGAAATGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAATGAAGAGAATCGAAGAAGGAATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACACCCGGTCGAAAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCTGCAGAACGGAAGAGACATGTACGTCGACCAGGAACTGGACATCAACAGACTGAGCGACTACGACGTCGACCACATCGTCCCGCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTCCTGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGTCCCGAGCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGACAGCTGCTGAACGCAAAGCTGATCACACAGAGAAAGTTCGACAACCTGACAAAGGCAGAGAGAGGAGGACTGAGCGAACTGGACAAGGCAGGATTCATCAAGAGACAGCTGGTCGAAACAAGACAGATCACAAAGCACGTCGCACAGATCCTGGACAGCAGAATGAACACAAAGTACGACGAAAACGACAAGCTGATCAGAGAAGTCAAGGTCATCACACTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGACTTCCAGTTCTACAAGGTCAGAGAAATCAACAACTACCACCACGCACACGACGCATACCTGAACGCAGTCGTCGGAACAGCACTGATCAAGAAGTACCCGAAGCTGGAAAGCGAATTCGTCTACGGAGACTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGAGCGAACAGGAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAAGACAGAAATCACACTGGCAAACGGAGAAATCAGAAAGAGACCGCTGATCGAAACAAACGGAGAAACAGGAGAAATCGTCTGGGACAAGGGAAGAGACTTCGCAACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCGTCAAGAAGACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAGCATCCTGCCGAAGAGAAACAGCGACAAGCTGATCGCAAGAAAGAAGGACTGGGACCCGAAGAAGTACGGAGGATTCGACAGCCCGACAGTCGCATACAGCGTCCTGGTCGTCGCAAAGGTCGAAAAGGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGCTGGGAATCACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGATCGACTTCCTGGAAGCAAAGGGATACAAGGAAGTCAAGAAGGACCTGATCATCAAGCTGCCGAAGTACAGCCTGTTCGAACTGGAAAACGGAAGAAAGAGAATGCTGGCAAGCGCAGGAGAACTGCAGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGTCAACTTCCTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCTGTTCGTCGAACAGCACAAGCACTACCTGGACGAAATCATCGAACAGATCAGCGAATTCAGCAAGAGAGTCATCCTGGCAGACGCAAACCTGGACAAGGTCCTGAGCGCATACAACAAGCACAGAGACAAGCCGATCAGAGAACAGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTGGGAGCACCGGCAGCATTCAAGTACTTCGACACAACAATCGACAGAAAGAGATACACAAGCACAAAGGAAGTCCTGGACGCAACACTGATCCACCAGAGCATCACAGGACTGTACGAAACAAGAATCGACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAAGCCCGAAGAAGAAGAGAAAGGTCTAGCTAGCCATCACATTTAAAAGCATCTCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAATAGCTTATTCATCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAACATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAATAAAAAATGGAAAGAACCTCGAG Cas9GGGACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACA 258 transcriptCCGGATCTGCCACCATGGACAAGAAGTACAGCATCGGACTGGACATCGGAA with 5′ UTRCAAACAGCGTCGGATGGGCAGTCATCACAGACGAATACAAGGTCCCGAGCA from HBB,AGAAGTTCAAGGTCCTGGGAAACACAGACAGACACAGCATCAAGAAGAACC ORFTGATCGGAGCACTGCTGTTCGACAGCGGAGAAACAGCAGAAGCAACAAGAC correspondingTGAAGAGAACAGCAAGAAGAAGATACACAAGAAGAAAGAACAGAATCTGCT to SEQ IDACCTGCAGGAAATCTTCAGCAACGAAATGGCAAAGGTCGACGACAGCTTCT NO: 204,TCCACAGACTGGAAGAAAGCTTCCTGGTCGAAGAAGACAAGAAGCACGAAA KozakGACACCCGATCTTCGGAAACATCGTCGACGAAGTCGCATACCACGAAAAGT sequence,ACCCGACAATCTACCACCTGAGAAAGAAGCTGGTCGACAGCACAGACAAGG and 3′ UTRCAGACCTGAGACTGATCTACCTGGCACTGGCACACATGATCAAGTTCAGAG of HBBGACACTTCCTGATCGAAGGAGACCTGAACCCGGACAACAGCGACGTCGACAAGCTGTTCATCCAGCTGGTCCAGACATACAACCAGCTGTTCGAAGAAAACCCGATCAACGCAAGCGGAGTCGACGCAAAGGCAATCCTGAGCGCAAGACTGAGCAAGAGCAGAAGACTGGAAAACCTGATCGCACAGCTGCCGGGAGAAAAGAAGAACGGACTGTTCGGAAACCTGATCGCACTGAGCCTGGGACTGACACCGAACTTCAAGAGCAACTTCGACCTGGCAGAAGACGCAAAGCTGCAGCTGAGCAAGGACACATACGACGACGACCTGGACAACCTGCTGGCACAGATCGGAGACCAGTACGCAGACCTGTTCCTGGCAGCAAAGAACCTGAGCGACGCAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAAATCACAAAGGCACCGCTGAGCGCAAGCATGATCAAGAGATACGACGAACACCACCAGGACCTGACACTGCTGAAGGCACTGGTCAGACAGCAGCTGCCGGAAAAGTACAAGGAAATCTTCTTCGACCAGAGCAAGAACGGATACGCAGGATACATCGACGGAGGAGCAAGCCAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAAGATGGACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAAGACCTGCTGAGAAAGCAGAGAACATTCGACAACGGAAGCATCCCGCACCAGATCCACCTGGGAGAACTGCACGCAATCCTGAGAAGACAGGAAGACTTCTACCCGTTCCTGAAGGACAACAGAGAAAAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTACGTCGGACCGCTGGCAAGAGGAAACAGCAGATTCGCATGGATGACAAGAAAGAGCGAAGAAACAATCACACCGTGGAACTTCGAAGAAGTCGTCGACAAGGGAGCAAGCGCACAGAGCTTCATCGAAAGAATGACAAACTTCGACAAGAACCTGCCGAACGAAAAGGTCCTGCCGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTACAACGAACTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCCGGCATTCCTGAGCGGAGAACAGAAGAAGGCAATCGTCGACCTGCTGTTCAAGACAAACAGAAAGGTCACAGTCAAGCAGCTGAAGGAAGACTACTTCAAGAAGATCGAATGCTTCGACAGCGTCGAAATCAGCGGAGTCGAAGACAGATTCAACGCAAGCCTGGGAACATACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGACAACGAAGAAAACGAAGACATCCTGGAAGACATCGTCCTGACACTGACACTGTTCGAAGACAGAGAAATGATCGAAGAAAGACTGAAGACATACGCACACCTGTTCGACGACAAGGTCATGAAGCAGCTGAAGAGAAGAAGATACACAGGATGGGGAAGACTGAGCAGAAAGCTGATCAACGGAATCAGAGACAAGCAGAGCGGAAAGACAATCCTGGACTTCCTGAAGAGCGACGGATTCGCAAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACATTCAAGGAAGACATCCAGAAGGCACAGGTCAGCGGACAGGGAGACAGCCTGCACGAACACATCGCAAACCTGGCAGGAAGCCCGGCAATCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAACTGGTCAAGGTCATGGGAAGACACAAGCCGGAAAACATCGTCATCGAAATGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAATGAAGAGAATCGAAGAAGGAATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACACCCGGTCGAAAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCTGCAGAACGGAAGAGACATGTACGTCGACCAGGAACTGGACATCAACAGACTGAGCGACTACGACGTCGACCACATCGTCCCGCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTCCTGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGTCCCGAGCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGACAGCTGCTGAACGCAAAGCTGATCACACAGAGAAAGTTCGACAACCTGACAAAGGCAGAGAGAGGAGGACTGAGCGAACTGGACAAGGCAGGATTCATCAAGAGACAGCTGGTCGAAACAAGACAGATCACAAAGCACGTCGCACAGATCCTGGACAGCAGAATGAACACAAAGTACGACGAAAACGACAAGCTGATCAGAGAAGTCAAGGTCATCACACTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGACTTCCAGTTCTACAAGGTCAGAGAAATCAACAACTACCACCACGCACACGACGCATACCTGAACGCAGTCGTCGGAACAGCACTGATCAAGAAGTACCCGAAGCTGGAAAGCGAATTCGTCTACGGAGACTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGAGCGAACAGGAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAAGACAGAAATCACACTGGCAAACGGAGAAATCAGAAAGAGACCGCTGATCGAAACAAACGGAGAAACAGGAGAAATCGTCTGGGACAAGGGAAGAGACTTCGCAACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCGTCAAGAAGACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAGCATCCTGCCGAAGAGAAACAGCGACAAGCTGATCGCAAGAAAGAAGGACTGGGACCCGAAGAAGTACGGAGGATTCGACAGCCCGACAGTCGCATACAGCGTCCTGGTCGTCGCAAAGGTCGAAAAGGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGCTGGGAATCACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGATCGACTTCCTGGAAGCAAAGGGATACAAGGAAGTCAAGAAGGACCTGATCATCAAGCTGCCGAAGTACAGCCTGTTCGAACTGGAAAACGGAAGAAAGAGAATGCTGGCAAGCGCAGGAGAACTGCAGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGTCAACTTCCTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCTGTTCGTCGAACAGCACAAGCACTACCTGGACGAAATCATCGAACAGATCAGCGAATTCAGCAAGAGAGTCATCCTGGCAGACGCAAACCTGGACAAGGTCCTGAGCGCATACAACAAGCACAGAGACAAGCCGATCAGAGAACAGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTGGGAGCACCGGCAGCATTCAAGTACTTCGACACAACAATCGACAGAAAGAGATACACAAGCACAAAGGAAGTCCTGGACGCAACACTGATCCACCAGAGCATCACAGGACTGTACGAAACAAGAATCGACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAAGCCCGAAGAAGAAGAGAAAGGTCTAGCTAGCGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTT TCATTGCCTCGAG Cas9GGGAAGCTCAGAATAAACGCTCAACTTTGGCCGGATCTGCCACCATGGACA 259 transcriptAGAAGTACAGCATCGGACTGGACATCGGAACAAACAGCGTCGGATGGGCAG with 5′ UTRTCATCACAGACGAATACAAGGTCCCGAGCAAGAAGTTCAAGGTCCTGGGAA from XBG,ACACAGACAGACACAGCATCAAGAAGAACCTGATCGGAGCACTGCTGTTCG ORFACAGCGGAGAAACAGCAGAAGCAACAAGACTGAAGAGAACAGCAAGAAGAA correspondingGATACACAAGAAGAAAGAACAGAATCTGCTACCTGCAGGAAATCTTCAGCA to SEQ IDACGAAATGGCAAAGGTCGACGACAGCTTCTTCCACAGACTGGAAGAAAGCT NO: 204,TCCTGGTCGAAGAAGACAAGAAGCACGAAAGACACCCGATCTTCGGAAACA KozakTCGTCGACGAAGTCGCATACCACGAAAAGTACCCGACAATCTACCACCTGA sequence,GAAAGAAGCTGGTCGACAGCACAGACAAGGCAGACCTGAGACTGATCTACC and 3′ UTRTGGCACTGGCACACATGATCAAGTTCAGAGGACACTTCCTGATCGAAGGAG of XBGACCTGAACCCGGACAACAGCGACGTCGACAAGCTGTTCATCCAGCTGGTCCAGACATACAACCAGCTGTTCGAAGAAAACCCGATCAACGCAAGCGGAGTCGACGCAAAGGCAATCCTGAGCGCAAGACTGAGCAAGAGCAGAAGACTGGAAAACCTGATCGCACAGCTGCCGGGAGAAAAGAAGAACGGACTGTTCGGAAACCTGATCGCACTGAGCCTGGGACTGACACCGAACTTCAAGAGCAACTTCGACCTGGCAGAAGACGCAAAGCTGCAGCTGAGCAAGGACACATACGACGACGACCTGGACAACCTGCTGGCACAGATCGGAGACCAGTACGCAGACCTGTTCCTGGCAGCAAAGAACCTGAGCGACGCAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAAATCACAAAGGCACCGCTGAGCGCAAGCATGATCAAGAGATACGACGAACACCACCAGGACCTGACACTGCTGAAGGCACTGGTCAGACAGCAGCTGCCGGAAAAGTACAAGGAAATCTTCTTCGACCAGAGCAAGAACGGATACGCAGGATACATCGACGGAGGAGCAAGCCAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAAGATGGACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAAGACCTGCTGAGAAAGCAGAGAACATTCGACAACGGAAGCATCCCGCACCAGATCCACCTGGGAGAACTGCACGCAATCCTGAGAAGACAGGAAGACTTCTACCCGTTCCTGAAGGACAACAGAGAAAAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTACGTCGGACCGCTGGCAAGAGGAAACAGCAGATTCGCATGGATGACAAGAAAGAGCGAAGAAACAATCACACCGTGGAACTTCGAAGAAGTCGTCGACAAGGGAGCAAGCGCACAGAGCTTCATCGAAAGAATGACAAACTTCGACAAGAACCTGCCGAACGAAAAGGTCCTGCCGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTACAACGAACTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCCGGCATTCCTGAGCGGAGAACAGAAGAAGGCAATCGTCGACCTGCTGTTCAAGACAAACAGAAAGGTCACAGTCAAGCAGCTGAAGGAAGACTACTTCAAGAAGATCGAATGCTTCGACAGCGTCGAAATCAGCGGAGTCGAAGACAGATTCAACGCAAGCCTGGGAACATACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGACAACGAAGAAAACGAAGACATCCTGGAAGACATCGTCCTGACACTGACACTGTTCGAAGACAGAGAAATGATCGAAGAAAGACTGAAGACATACGCACACCTGTTCGACGACAAGGTCATGAAGCAGCTGAAGAGAAGAAGATACACAGGATGGGGAAGACTGAGCAGAAAGCTGATCAACGGAATCAGAGACAAGCAGAGCGGAAAGACAATCCTGGACTTCCTGAAGAGCGACGGATTCGCAAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACATTCAAGGAAGACATCCAGAAGGCACAGGTCAGCGGACAGGGAGACAGCCTGCACGAACACATCGCAAACCTGGCAGGAAGCCCGGCAATCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAACTGGTCAAGGTCATGGGAAGACACAAGCCGGAAAACATCGTCATCGAAATGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAATGAAGAGAATCGAAGAAGGAATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACACCCGGTCGAAAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCTGCAGAACGGAAGAGACATGTACGTCGACCAGGAACTGGACATCAACAGACTGAGCGACTACGACGTCGACCACATCGTCCCGCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTCCTGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGTCCCGAGCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGACAGCTGCTGAACGCAAAGCTGATCACACAGAGAAAGTTCGACAACCTGACAAAGGCAGAGAGAGGAGGACTGAGCGAACTGGACAAGGCAGGATTCATCAAGAGACAGCTGGTCGAAACAAGACAGATCACAAAGCACGTCGCACAGATCCTGGACAGCAGAATGAACACAAAGTACGACGAAAACGACAAGCTGATCAGAGAAGTCAAGGTCATCACACTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGACTTCCAGTTCTACAAGGTCAGAGAAATCAACAACTACCACCACGCACACGACGCATACCTGAACGCAGTCGTCGGAACAGCACTGATCAAGAAGTACCCGAAGCTGGAAAGCGAATTCGTCTACGGAGACTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGAGCGAACAGGAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAAGACAGAAATCACACTGGCAAACGGAGAAATCAGAAAGAGACCGCTGATCGAAACAAACGGAGAAACAGGAGAAATCGTCTGGGACAAGGGAAGAGACTTCGCAACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCGTCAAGAAGACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAGCATCCTGCCGAAGAGAAACAGCGACAAGCTGATCGCAAGAAAGAAGGACTGGGACCCGAAGAAGTACGGAGGATTCGACAGCCCGACAGTCGCATACAGCGTCCTGGTCGTCGCAAAGGTCGAAAAGGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGCTGGGAATCACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGATCGACTTCCTGGAAGCAAAGGGATACAAGGAAGTCAAGAAGGACCTGATCATCAAGCTGCCGAAGTACAGCCTGTTCGAACTGGAAAACGGAAGAAAGAGAATGCTGGCAAGCGCAGGAGAACTGCAGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGTCAACTTCCTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCTGTTCGTCGAACAGCACAAGCACTACCTGGACGAAATCATCGAACAGATCAGCGAATTCAGCAAGAGAGTCATCCTGGCAGACGCAAACCTGGACAAGGTCCTGAGCGCATACAACAAGCACAGAGACAAGCCGATCAGAGAACAGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTGGGAGCACCGGCAGCATTCAAGTACTTCGACACAACAATCGACAGAAAGAGATACACAAGCACAAAGGAAGTCCTGGACGCAACACTGATCCACCAGAGCATCACAGGACTGTACGAAACAAGAATCGACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAAGCCCGAAGAAGAAGAGAAAGGTCTAGCTAGCACCAGCCTCAAGAACACCCGAATGGAGTCTCTAAGCTACATAATACCAACTTACACTTTACAAAATGTTGTCCCCCAAAATGTAGCCATTCGTATCTGCTCCTAATAAAAAGAAAGTTTCTTCACATTCTCTCGAG Cas9AGGAAGCTCAGAATAAACGCTCAACTTTGGCCGGATCTGCCACCATGGACA 260 transcriptAGAAGTACAGCATCGGACTGGACATCGGAACAAACAGCGTCGGATGGGCAG with AGG asTCATCACAGACGAATACAAGGTCCCGAGCAAGAAGTTCAAGGTCCTGGGAA first threeACACAGACAGACACAGCATCAAGAAGAACCTGATCGGAGCACTGCTGTTCG nucleotidesACAGCGGAGAAACAGCAGAAGCAACAAGACTGAAGAGAACAGCAAGAAGAA for use withGATACACAAGAAGAAAGAACAGAATCTGCTACCTGCAGGAAATCTTCAGCA CleanCap ™,ACGAAATGGCAAAGGTCGACGACAGCTTCTTCCACAGACTGGAAGAAAGCT 5′ UTR fromTCCTGGTCGAAGAAGACAAGAAGCACGAAAGACACCCGATCTTCGGAAACA XBG, ORFTCGTCGACGAAGTCGCATACCACGAAAAGTACCCGACAATCTACCACCTGA correspondingGAAAGAAGCTGGTCGACAGCACAGACAAGGCAGACCTGAGACTGATCTACC to SEQ IDTGGCACTGGCACACATGATCAAGTTCAGAGGACACTTCCTGATCGAAGGAG NO: 204,ACCTGAACCCGGACAACAGCGACGTCGACAAGCTGTTCATCCAGCTGGTCC KozakAGACATACAACCAGCTGTTCGAAGAAAACCCGATCAACGCAAGCGGAGTCG sequence,ACGCAAAGGCAATCCTGAGCGCAAGACTGAGCAAGAGCAGAAGACTGGAAA and 3′ UTRACCTGATCGCACAGCTGCCGGGAGAAAAGAAGAACGGACTGTTCGGAAACC of XBGTGATCGCACTGAGCCTGGGACTGACACCGAACTTCAAGAGCAACTTCGACCTGGCAGAAGACGCAAAGCTGCAGCTGAGCAAGGACACATACGACGACGACCTGGACAACCTGCTGGCACAGATCGGAGACCAGTACGCAGACCTGTTCCTGGCAGCAAAGAACCTGAGCGACGCAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAAATCACAAAGGCACCGCTGAGCGCAAGCATGATCAAGAGATACGACGAACACCACCAGGACCTGACACTGCTGAAGGCACTGGTCAGACAGCAGCTGCCGGAAAAGTACAAGGAAATCTTCTTCGACCAGAGCAAGAACGGATACGCAGGATACATCGACGGAGGAGCAAGCCAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAAGATGGACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAAGACCTGCTGAGAAAGCAGAGAACATTCGACAACGGAAGCATCCCGCACCAGATCCACCTGGGAGAACTGCACGCAATCCTGAGAAGACAGGAAGACTTCTACCCGTTCCTGAAGGACAACAGAGAAAAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTACGTCGGACCGCTGGCAAGAGGAAACAGCAGATTCGCATGGATGACAAGAAAGAGCGAAGAAACAATCACACCGTGGAACTTCGAAGAAGTCGTCGACAAGGGAGCAAGCGCACAGAGCTTCATCGAAAGAATGACAAACTTCGACAAGAACCTGCCGAACGAAAAGGTCCTGCCGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTACAACGAACTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCCGGCATTCCTGAGCGGAGAACAGAAGAAGGCAATCGTCGACCTGCTGTTCAAGACAAACAGAAAGGTCACAGTCAAGCAGCTGAAGGAAGACTACTTCAAGAAGATCGAATGCTTCGACAGCGTCGAAATCAGCGGAGTCGAAGACAGATTCAACGCAAGCCTGGGAACATACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGACAACGAAGAAAACGAAGACATCCTGGAAGACATCGTCCTGACACTGACACTGTTCGAAGACAGAGAAATGATCGAAGAAAGACTGAAGACATACGCACACCTGTTCGACGACAAGGTCATGAAGCAGCTGAAGAGAAGAAGATACACAGGATGGGGAAGACTGAGCAGAAAGCTGATCAACGGAATCAGAGACAAGCAGAGCGGAAAGACAATCCTGGACTTCCTGAAGAGCGACGGATTCGCAAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACATTCAAGGAAGACATCCAGAAGGCACAGGTCAGCGGACAGGGAGACAGCCTGCACGAACACATCGCAAACCTGGCAGGAAGCCCGGCAATCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAACTGGTCAAGGTCATGGGAAGACACAAGCCGGAAAACATCGTCATCGAAATGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAATGAAGAGAATCGAAGAAGGAATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACACCCGGTCGAAAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCTGCAGAACGGAAGAGACATGTACGTCGACCAGGAACTGGACATCAACAGACTGAGCGACTACGACGTCGACCACATCGTCCCGCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTCCTGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGTCCCGAGCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGACAGCTGCTGAACGCAAAGCTGATCACACAGAGAAAGTTCGACAACCTGACAAAGGCAGAGAGAGGAGGACTGAGCGAACTGGACAAGGCAGGATTCATCAAGAGACAGCTGGTCGAAACAAGACAGATCACAAAGCACGTCGCACAGATCCTGGACAGCAGAATGAACACAAAGTACGACGAAAACGACAAGCTGATCAGAGAAGTCAAGGTCATCACACTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGACTTCCAGTTCTACAAGGTCAGAGAAATCAACAACTACCACCACGCACACGACGCATACCTGAACGCAGTCGTCGGAACAGCACTGATCAAGAAGTACCCGAAGCTGGAAAGCGAATTCGTCTACGGAGACTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGAGCGAACAGGAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAAGACAGAAATCACACTGGCAAACGGAGAAATCAGAAAGAGACCGCTGATCGAAACAAACGGAGAAACAGGAGAAATCGTCTGGGACAAGGGAAGAGACTTCGCAACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCGTCAAGAAGACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAGCATCCTGCCGAAGAGAAACAGCGACAAGCTGATCGCAAGAAAGAAGGACTGGGACCCGAAGAAGTACGGAGGATTCGACAGCCCGACAGTCGCATACAGCGTCCTGGTCGTCGCAAAGGTCGAAAAGGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGCTGGGAATCACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGATCGACTTCCTGGAAGCAAAGGGATACAAGGAAGTCAAGAAGGACCTGATCATCAAGCTGCCGAAGTACAGCCTGTTCGAACTGGAAAACGGAAGAAAGAGAATGCTGGCAAGCGCAGGAGAACTGCAGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGTCAACTTCCTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCTGTTCGTCGAACAGCACAAGCACTACCTGGACGAAATCATCGAACAGATCAGCGAATTCAGCAAGAGAGTCATCCTGGCAGACGCAAACCTGGACAAGGTCCTGAGCGCATACAACAAGCACAGAGACAAGCCGATCAGAGAACAGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTGGGAGCACCGGCAGCATTCAAGTACTTCGACACAACAATCGACAGAAAGAGATACACAAGCACAAAGGAAGTCCTGGACGCAACACTGATCCACCAGAGCATCACAGGACTGTACGAAACAAGAATCGACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAAGCCCGAAGAAGAAGAGAAAGGTCTAGCTAGCACCAGCCTCAAGAACACCCGAATGGAGTCTCTAAGCTACATAATACCAACTTACACTTTACAAAATGTTGTCCCCCAAAATGTAGCCATTCGTATCTGCTCCTAATAAAAAGAAAGTTTCTTCACATTCTCTCGAG Cas9AGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGTGTGTCGTT 261 transcriptGCAGGCCTTATTCGGATCCGCCACCATGGACAAGAAGTACAGCATCGGACT with AGG asGGACATCGGAACAAACAGCGTCGGATGGGCAGTCATCACAGACGAATACAA first threeGGTCCCGAGCAAGAAGTTCAAGGTCCTGGGAAACACAGACAGACACAGCAT nucleotidesCAAGAAGAACCTGATCGGAGCACTGCTGTTCGACAGCGGAGAAACAGCAGA for use withAGCAACAAGACTGAAGAGAACAGCAAGAAGAAGATACACAAGAAGAAAGAA CleanCap ™,CAGAATCTGCTACCTGCAGGAAATCTTCAGCAACGAAATGGCAAAGGTCGA 5′ UTR fromCGACAGCTTCTTCCACAGACTGGAAGAAAGCTTCCTGGTCGAAGAAGACAA HSD, ORFGAAGCACGAAAGACACCCGATCTTCGGAAACATCGTCGACGAAGTCGCATA correspondingCCACGAAAAGTACCCGACAATCTACCACCTGAGAAAGAAGCTGGTCGACAG to SEQ IDCACAGACAAGGCAGACCTGAGACTGATCTACCTGGCACTGGCACACATGAT NO: 204,CAAGTTCAGAGGACACTTCCTGATCGAAGGAGACCTGAACCCGGACAACAG KozakCGACGTCGACAAGCTGTTCATCCAGCTGGTCCAGACATACAACCAGCTGTT sequence,CGAAGAAAACCCGATCAACGCAAGCGGAGTCGACGCAAAGGCAATCCTGAG and 3′ UTRCGCAAGACTGAGCAAGAGCAGAAGACTGGAAAACCTGATCGCACAGCTGCC of ALBGGGAGAAAAGAAGAACGGACTGTTCGGAAACCTGATCGCACTGAGCCTGGGACTGACACCGAACTTCAAGAGCAACTTCGACCTGGCAGAAGACGCAAAGCTGCAGCTGAGCAAGGACACATACGACGACGACCTGGACAACCTGCTGGCACAGATCGGAGACCAGTACGCAGACCTGTTCCTGGCAGCAAAGAACCTGAGCGACGCAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAAATCACAAAGGCACCGCTGAGCGCAAGCATGATCAAGAGATACGACGAACACCACCAGGACCTGACACTGCTGAAGGCACTGGTCAGACAGCAGCTGCCGGAAAAGTACAAGGAAATCTTCTTCGACCAGAGCAAGAACGGATACGCAGGATACATCGACGGAGGAGCAAGCCAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAAGATGGACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAAGACCTGCTGAGAAAGCAGAGAACATTCGACAACGGAAGCATCCCGCACCAGATCCACCTGGGAGAACTGCACGCAATCCTGAGAAGACAGGAAGACTTCTACCCGTTCCTGAAGGACAACAGAGAAAAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTACGTCGGACCGCTGGCAAGAGGAAACAGCAGATTCGCATGGATGACAAGAAAGAGCGAAGAAACAATCACACCGTGGAACTTCGAAGAAGTCGTCGACAAGGGAGCAAGCGCACAGAGCTTCATCGAAAGAATGACAAACTTCGACAAGAACCTGCCGAACGAAAAGGTCCTGCCGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTACAACGAACTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCCGGCATTCCTGAGCGGAGAACAGAAGAAGGCAATCGTCGACCTGCTGTTCAAGACAAACAGAAAGGTCACAGTCAAGCAGCTGAAGGAAGACTACTTCAAGAAGATCGAATGCTTCGACAGCGTCGAAATCAGCGGAGTCGAAGACAGATTCAACGCAAGCCTGGGAACATACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGACAACGAAGAAAACGAAGACATCCTGGAAGACATCGTCCTGACACTGACACTGTTCGAAGACAGAGAAATGATCGAAGAAAGACTGAAGACATACGCACACCTGTTCGACGACAAGGTCATGAAGCAGCTGAAGAGAAGAAGATACACAGGATGGGGAAGACTGAGCAGAAAGCTGATCAACGGAATCAGAGACAAGCAGAGCGGAAAGACAATCCTGGACTTCCTGAAGAGCGACGGATTCGCAAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACATTCAAGGAAGACATCCAGAAGGCACAGGTCAGCGGACAGGGAGACAGCCTGCACGAACACATCGCAAACCTGGCAGGAAGCCCGGCAATCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAACTGGTCAAGGTCATGGGAAGACACAAGCCGGAAAACATCGTCATCGAAATGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAATGAAGAGAATCGAAGAAGGAATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACACCCGGTCGAAAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCTGCAGAACGGAAGAGACATGTACGTCGACCAGGAACTGGACATCAACAGACTGAGCGACTACGACGTCGACCACATCGTCCCGCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTCCTGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGTCCCGAGCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGACAGCTGCTGAACGCAAAGCTGATCACACAGAGAAAGTTCGACAACCTGACAAAGGCAGAGAGAGGAGGACTGAGCGAACTGGACAAGGCAGGATTCATCAAGAGACAGCTGGTCGAAACAAGACAGATCACAAAGCACGTCGCACAGATCCTGGACAGCAGAATGAACACAAAGTACGACGAAAACGACAAGCTGATCAGAGAAGTCAAGGTCATCACACTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGACTTCCAGTTCTACAAGGTCAGAGAAATCAACAACTACCACCACGCACACGACGCATACCTGAACGCAGTCGTCGGAACAGCACTGATCAAGAAGTACCCGAAGCTGGAAAGCGAATTCGTCTACGGAGACTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGAGCGAACAGGAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAAGACAGAAATCACACTGGCAAACGGAGAAATCAGAAAGAGACCGCTGATCGAAACAAACGGAGAAACAGGAGAAATCGTCTGGGACAAGGGAAGAGACTTCGCAACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCGTCAAGAAGACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAGCATCCTGCCGAAGAGAAACAGCGACAAGCTGATCGCAAGAAAGAAGGACTGGGACCCGAAGAAGTACGGAGGATTCGACAGCCCGACAGTCGCATACAGCGTCCTGGTCGTCGCAAAGGTCGAAAAGGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGCTGGGAATCACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGATCGACTTCCTGGAAGCAAAGGGATACAAGGAAGTCAAGAAGGACCTGATCATCAAGCTGCCGAAGTACAGCCTGTTCGAACTGGAAAACGGAAGAAAGAGAATGCTGGCAAGCGCAGGAGAACTGCAGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGTCAACTTCCTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCTGTTCGTCGAACAGCACAAGCACTACCTGGACGAAATCATCGAACAGATCAGCGAATTCAGCAAGAGAGTCATCCTGGCAGACGCAAACCTGGACAAGGTCCTGAGCGCATACAACAAGCACAGAGACAAGCCGATCAGAGAACAGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTGGGAGCACCGGCAGCATTCAAGTACTTCGACACAACAATCGACAGAAAGAGATACACAAGCACAAAGGAAGTCCTGGACGCAACACTGATCCACCAGAGCATCACAGGACTGTACGAAACAAGAATCGACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAAGCCCGAAGAAGAAGAGAAAGGTCTAGCTAGCCATCACATTTAAAAGCATCTCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAATAGCTTATTCATCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAACATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAATAAAAAATGGAAAGAACCTCGAG 30/30/39 Not used 262 poly-A sequencepoly-A 100 AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 263sequence AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA G209 singleAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACC 264 guide RNA targetingthe mouse TTR gene ORF encodingATGGCAGCATTCAAGCCGAACTCGATCAACTACATCCTGGGACTGGACATC 265 NeisseriaGGAATCGCATCGGTCGGATGGGCAATGGTCGAAATCGACGAAGAAGAAAAC meningitidisCCGATCAGACTGATCGACCTGGGAGTCAGAGTCTTCGAAAGAGCAGAAGTC Cas9 usingCCGAAGACAGGAGACTCGCTGGCAATGGCAAGAAGACTGGCAAGATCGGTC minimalAGAAGACTGACAAGAAGAAGAGCACACAGACTGCTGAGAACAAGAAGACTG uridineCTGAAGAGAGAAGGAGTCCTGCAGGCAGCAAACTTCGACGAAAACGGACTG codons, withATCAAGTCGCTGCCGAACACACCGTGGCAGCTGAGAGCAGCAGCACTGGAC start andAGAAAGCTGACACCGCTGGAATGGTCGGCAGTCCTGCTGCACCTGATCAAG stop codonsCACAGAGGATACCTGTCGCAGAGAAAGAACGAAGGAGAAACAGCAGACAAGGAACTGGGAGCACTGCTGAAGGGAGTCGCAGGAAACGCACACGCACTGCAGACAGGAGACTTCAGAACACCGGCAGAACTGGCACTGAACAAGTTCGAAAAGGAATCGGGACACATCAGAAACCAGAGATCGGACTACTCGCACACATTCTCGAGAAAGGACCTGCAGGCAGAACTGATCCTGCTGTTCGAAAAGCAGAAGGAATTCGGAAACCCGCACGTCTCGGGAGGACTGAAGGAAGGAATCGAAACACTGCTGATGACACAGAGACCGGCACTGTCGGGAGACGCAGTCCAGAAGATGCTGGGACACTGCACATTCGAACCGGCAGAACCGAAGGCAGCAAAGAACACATACACAGCAGAAAGATTCATCTGGCTGACAAAGCTGAACAACCTGAGAATCCTGGAACAGGGATCGGAAAGACCGCTGACAGACACAGAAAGAGCAACACTGATGGACGAACCGTACAGAAAGTCGAAGCTGACATACGCACAGGCAAGAAAGCTGCTGGGACTGGAAGACACAGCATTCTTCAAGGGACTGAGATACGGAAAGGACAACGCAGAAGCATCGACACTGATGGAAATGAAGGCATACCACGCAATCTCGAGAGCACTGGAAAAGGAAGGACTGAAGGACAAGAAGTCGCCGCTGAACCTGTCGCCGGAACTGCAGGACGAAATCGGAACAGCATTCTCGCTGTTCAAGACAGACGAAGACATCACAGGAAGACTGAAGGACAGAATCCAGCCGGAAATCCTGGAAGCACTGCTGAAGCACATCTCGTTCGACAAGTTCGTCCAGATCTCGCTGAAGGCACTGAGAAGAATCGTCCCGCTGATGGAACAGGGAAAGAGATACGACGAAGCATGCGCAGAAATCTACGGAGACCACTACGGAAAGAAGAACACAGAAGAAAAGATCTACCTGCCGCCGATCCCGGCAGACGAAATCAGAAACCCGGTCGTCCTGAGAGCACTGTCGCAGGCAAGAAAGGTCATCAACGGAGTCGTCAGAAGATACGGATCGCCGGCAAGAATCCACATCGAAACAGCAAGAGAAGTCGGAAAGTCGTTCAAGGACAGAAAGGAAATCGAAAAGAGACAGGAAGAAAACAGAAAGGACAGAGAAAAGGCAGCAGCAAAGTTCAGAGAATACTTCCCGAACTTCGTCGGAGAACCGAAGTCGAAGGACATCCTGAAGCTGAGACTGTACGAACAGCAGCACGGAAAGTGCCTGTACTCGGGAAAGGAAATCAACCTGGGAAGACTGAACGAAAAGGGATACGTCGAAATCGACCACGCACTGCCGTTCTCGAGAACATGGGACGACTCGTTCAACAACAAGGTCCTGGTCCTGGGATCGGAAAACCAGAACAAGGGAAACCAGACACCGTACGAATACTTCAACGGAAAGGACAACTCGAGAGAATGGCAGGAATTCAAGGCAAGAGTCGAAACATCGAGATTCCCGAGATCGAAGAAGCAGAGAATCCTGCTGCAGAAGTTCGACGAAGACGGATTCAAGGAAAGAAACCTGAACGACACAAGATACGTCAACAGATTCCTGTGCCAGTTCGTCGCAGACAGAATGAGACTGACAGGAAAGGGAAAGAAGAGAGTCTTCGCATCGAACGGACAGATCACAAACCTGCTGAGAGGATTCTGGGGACTGAGAAAGGTCAGAGCAGAAAACGACAGACACCACGCACTGGACGCAGTCGTCGTCGCATGCTCGACAGTCGCAATGCAGCAGAAGATCACAAGATTCGTCAGATACAAGGAAATGAACGCATTCGACGGAAAGACAATCGACAAGGAAACAGGAGAAGTCCTGCACCAGAAGACACACTTCCCGCAGCCGTGGGAATTCTTCGCACAGGAAGTCATGATCAGAGTCTTCGGAAAGCCGGACGGAAAGCCGGAATTCGAAGAAGCAGACACACTGGAAAAGCTGAGAACACTGCTGGCAGAAAAGCTGTCGTCGAGACCGGAAGCAGTCCACGAATACGTCACACCGCTGTTCGTCTCGAGAGCACCGAACAGAAAGATGTCGGGACAGGGACACATGGAAACAGTCAAGTCGGCAAAGAGACTGGACGAAGGAGTCTCGGTCCTGAGAGTCCCGCTGACACAGCTGAAGCTGAAGGACCTGGAAAAGATGGTCAACAGAGAAAGAGAACCGAAGCTGTACGAAGCACTGAAGGCAAGACTGGAAGCACACAAGGACGACCCGGCAAAGGCATTCGCAGAACCGTTCTACAAGTACGACAAGGCAGGAAACAGAACACAGCAGGTCAAGGCAGTCAGAGTCGAACAGGTCCAGAAGACAGGAGTCTGGGTCAGAAACCACAACGGAATCGCAGACAACGCAACAATGGTCAGAGTAGACGTCTTCGAAAAGGGAGACAAGTACTACCTGGTCCCGATCTACTCGTGGCAGGTCGCAAAGGGAATCCTGCCGGACAGAGCAGTCGTCCAGGGAAAGGACGAAGAAGACTGGCAGCTGATCGACGACTCGTTCAACTTCAAGTTCTCGCTGCACCCGAACGACCTGGTCGAAGTCATCACAAAGAAGGCAAGAATGTTCGGATACTTCGCATCGTGCCACAGAGGAACAGGAAACATCAACATCAGAATCCACGACCTGGACCACAAGATCGGAAAGAACGGAATCCTGGAAGGAATCGGAGTCAAGACAGCACTGTCGTTCCAGAAGTACCAGATCGACGAACTGGGAAAGGAAATCAGACCGTGCAGACTGAAGAAGAGACCGCCGGTCAGATCCGGAAAGAGAACAGCAGACGGATCGGAATTCGAATCGCCGAAGAAGAAGAGAAAGGTCGAATGA ORF encodingGCAGCATTCAAGCCGAACTCGATCAACTACATCCTGGGACTGGACATCGGA 266 NeisseriaATCGCATCGGTCGGATGGGCAATGGTCGAAATCGACGAAGAAGAAAACCCG meningitidisATCAGACTGATCGACCTGGGAGTCAGAGTCTTCGAAAGAGCAGAAGTCCCG Cas9 usingAAGACAGGAGACTCGCTGGCAATGGCAAGAAGACTGGCAAGATCGGTCAGA minimalAGACTGACAAGAAGAAGAGCACACAGACTGCTGAGAACAAGAAGACTGCTG uridineAAGAGAGAAGGAGTCCTGCAGGCAGCAAACTTCGACGAAAACGGACTGATC codons (noAAGTCGCTGCCGAACACACCGTGGCAGCTGAGAGCAGCAGCACTGGACAGA start orAAGCTGACACCGCTGGAATGGTCGGCAGTCCTGCTGCACCTGATCAAGCAC stop codons;AGAGGATACCTGTCGCAGAGAAAGAACGAAGGAGAAACAGCAGACAAGGAA suitable forCTGGGAGCACTGCTGAAGGGAGTCGCAGGAAACGCACACGCACTGCAGACA inclusion inGGAGACTTCAGAACACCGGCAGAACTGGCACTGAACAAGTTCGAAAAGGAA fusionTCGGGACACATCAGAAACCAGAGATCGGACTACTCGCACACATTCTCGAGA proteinAAGGACCTGCAGGCAGAACTGATCCTGCTGTTCGAAAAGCAGAAGGAATTC codingGGAAACCCGCACGTCTCGGGAGGACTGAAGGAAGGAATCGAAACACTGCTG sequence)ATGACACAGAGACCGGCACTGTCGGGAGACGCAGTCCAGAAGATGCTGGGACACTGCACATTCGAACCGGCAGAACCGAAGGCAGCAAAGAACACATACACAGCAGAAAGATTCATCTGGCTGACAAAGCTGAACAACCTGAGAATCCTGGAACAGGGATCGGAAAGACCGCTGACAGACACAGAAAGAGCAACACTGATGGACGAACCGTACAGAAAGTCGAAGCTGACATACGCACAGGCAAGAAAGCTGCTGGGACTGGAAGACACAGCATTCTTCAAGGGACTGAGATACGGAAAGGACAACGCAGAAGCATCGACACTGATGGAAATGAAGGCATACCACGCAATCTCGAGAGCACTGGAAAAGGAAGGACTGAAGGACAAGAAGTCGCCGCTGAACCTGTCGCCGGAACTGCAGGACGAAATCGGAACAGCATTCTCGCTGTTCAAGACAGACGAAGACATCACAGGAAGACTGAAGGACAGAATCCAGCCGGAAATCCTGGAAGCACTGCTGAAGCACATCTCGTTCGACAAGTTCGTCCAGATCTCGCTGAAGGCACTGAGAAGAATCGTCCCGCTGATGGAACAGGGAAAGAGATACGACGAAGCATGCGCAGAAATCTACGGAGACCACTACGGAAAGAAGAACACAGAAGAAAAGATCTACCTGCCGCCGATCCCGGCAGACGAAATCAGAAACCCGGTCGTCCTGAGAGCACTGTCGCAGGCAAGAAAGGTCATCAACGGAGTCGTCAGAAGATACGGATCGCCGGCAAGAATCCACATCGAAACAGCAAGAGAAGTCGGAAAGTCGTTCAAGGACAGAAAGGAAATCGAAAAGAGACAGGAAGAAAACAGAAAGGACAGAGAAAAGGCAGCAGCAAAGTTCAGAGAATACTTCCCGAACTTCGTCGGAGAACCGAAGTCGAAGGACATCCTGAAGCTGAGACTGTACGAACAGCAGCACGGAAAGTGCCTGTACTCGGGAAAGGAAATCAACCTGGGAAGACTGAACGAAAAGGGATACGTCGAAATCGACCACGCACTGCCGTTCTCGAGAACATGGGACGACTCGTTCAACAACAAGGTCCTGGTCCTGGGATCGGAAAACCAGAACAAGGGAAACCAGACACCGTACGAATACTTCAACGGAAAGGACAACTCGAGAGAATGGCAGGAATTCAAGGCAAGAGTCGAAACATCGAGATTCCCGAGATCGAAGAAGCAGAGAATCCTGCTGCAGAAGTTCGACGAAGACGGATTCAAGGAAAGAAACCTGAACGACACAAGATACGTCAACAGATTCCTGTGCCAGTTCGTCGCAGACAGAATGAGACTGACAGGAAAGGGAAAGAAGAGAGTCTTCGCATCGAACGGACAGATCACAAACCTGCTGAGAGGATTCTGGGGACTGAGAAAGGTCAGAGCAGAAAACGACAGACACCACGCACTGGACGCAGTCGTCGTCGCATGCTCGACAGTCGCAATGCAGCAGAAGATCACAAGATTCGTCAGATACAAGGAAATGAACGCATTCGACGGAAAGACAATCGACAAGGAAACAGGAGAAGTCCTGCACCAGAAGACACACTTCCCGCAGCCGTGGGAATTCTTCGCACAGGAAGTCATGATCAGAGTCTTCGGAAAGCCGGACGGAAAGCCGGAATTCGAAGAAGCAGACACACTGGAAAAGCTGAGAACACTGCTGGCAGAAAAGCTGTCGTCGAGACCGGAAGCAGTCCACGAATACGTCACACCGCTGTTCGTCTCGAGAGCACCGAACAGAAAGATGTCGGGACAGGGACACATGGAAACAGTCAAGTCGGCAAAGAGACTGGACGAAGGAGTCTCGGTCCTGAGAGTCCCGCTGACACAGCTGAAGCTGAAGGACCTGGAAAAGATGGTCAACAGAGAAAGAGAACCGAAGCTGTACGAAGCACTGAAGGCAAGACTGGAAGCACACAAGGACGACCCGGCAAAGGCATTCGCAGAACCGTTCTACAAGTACGACAAGGCAGGAAACAGAACACAGCAGGTCAAGGCAGTCAGAGTCGAACAGGTCCAGAAGACAGGAGTCTGGGTCAGAAACCACAACGGAATCGCAGACAACGCAACAATGGTCAGAGTAGACGTCTTCGAAAAGGGAGACAAGTACTACCTGGTCCCGATCTACTCGTGGCAGGTCGCAAAGGGAATCCTGCCGGACAGAGCAGTCGTCCAGGGAAAGGACGAAGAAGACTGGCAGCTGATCGACGACTCGTTCAACTTCAAGTTCTCGCTGCACCCGAACGACCTGGTCGAAGTCATCACAAAGAAGGCAAGAATGTTCGGATACTTCGCATCGTGCCACAGAGGAACAGGAAACATCAACATCAGAATCCACGACCTGGACCACAAGATCGGAAAGAACGGAATCCTGGAAGGAATCGGAGTCAAGACAGCACTGTCGTTCCAGAAGTACCAGATCGACGAACTGGGAAAGGAAATCAGACCGTGCAGACTGAAGAAGAGACCGCCGGTCAGATCCGGAAAGAGAACAGCAGACGGATCGGAATTCGAATCGCCGAAGAAGAAGAGAAAGGTCGAA TranscriptGGGAGACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTGGATCCGCCACCAT 267 comprisingGGCAGCATTCAAGCCGAACTCGATCAACTACATCCTGGGACTGGACATCGG SEQ ID NO:AATCGCATCGGTCGGATGGGCAATGGTCGAAATCGACGAAGAAGAAAACCC 265GATCAGACTGATCGACCTGGGAGTCAGAGTCTTCGAAAGAGCAGAAGTCCC (encodingGAAGACAGGAGACTCGCTGGCAATGGCAAGAAGACTGGCAAGATCGGTCAG NeisseriaAAGACTGACAAGAAGAAGAGCACACAGACTGCTGAGAACAAGAAGACTGCT meningitidisGAAGAGAGAAGGAGTCCTGCAGGCAGCAAACTTCGACGAAAACGGACTGAT Cas9)CAAGTCGCTGCCGAACACACCGTGGCAGCTGAGAGCAGCAGCACTGGACAGAAAGCTGACACCGCTGGAATGGTCGGCAGTCCTGCTGCACCTGATCAAGCACAGAGGATACCTGTCGCAGAGAAAGAACGAAGGAGAAACAGCAGACAAGGAACTGGGAGCACTGCTGAAGGGAGTCGCAGGAAACGCACACGCACTGCAGACAGGAGACTTCAGAACACCGGCAGAACTGGCACTGAACAAGTTCGAAAAGGAATCGGGACACATCAGAAACCAGAGATCGGACTACTCGCACACATTCTCGAGAAAGGACCTGCAGGCAGAACTGATCCTGCTGTTCGAAAAGCAGAAGGAATTCGGAAACCCGCACGTCTCGGGAGGACTGAAGGAAGGAATCGAAACACTGCTGATGACACAGAGACCGGCACTGTCGGGAGACGCAGTCCAGAAGATGCTGGGACACTGCACATTCGAACCGGCAGAACCGAAGGCAGCAAAGAACACATACACAGCAGAAAGATTCATCTGGCTGACAAAGCTGAACAACCTGAGAATCCTGGAACAGGGATCGGAAAGACCGCTGACAGACACAGAAAGAGCAACACTGATGGACGAACCGTACAGAAAGTCGAAGCTGACATACGCACAGGCAAGAAAGCTGCTGGGACTGGAAGACACAGCATTCTTCAAGGGACTGAGATACGGAAAGGACAACGCAGAAGCATCGACACTGATGGAAATGAAGGCATACCACGCAATCTCGAGAGCACTGGAAAAGGAAGGACTGAAGGACAAGAAGTCGCCGCTGAACCTGTCGCCGGAACTGCAGGACGAAATCGGAACAGCATTCTCGCTGTTCAAGACAGACGAAGACATCACAGGAAGACTGAAGGACAGAATCCAGCCGGAAATCCTGGAAGCACTGCTGAAGCACATCTCGTTCGACAAGTTCGTCCAGATCTCGCTGAAGGCACTGAGAAGAATCGTCCCGCTGATGGAACAGGGAAAGAGATACGACGAAGCATGCGCAGAAATCTACGGAGACCACTACGGAAAGAAGAACACAGAAGAAAAGATCTACCTGCCGCCGATCCCGGCAGACGAAATCAGAAACCCGGTCGTCCTGAGAGCACTGTCGCAGGCAAGAAAGGTCATCAACGGAGTCGTCAGAAGATACGGATCGCCGGCAAGAATCCACATCGAAACAGCAAGAGAAGTCGGAAAGTCGTTCAAGGACAGAAAGGAAATCGAAAAGAGACAGGAAGAAAACAGAAAGGACAGAGAAAAGGCAGCAGCAAAGTTCAGAGAATACTTCCCGAACTTCGTCGGAGAACCGAAGTCGAAGGACATCCTGAAGCTGAGACTGTACGAACAGCAGCACGGAAAGTGCCTGTACTCGGGAAAGGAAATCAACCTGGGAAGACTGAACGAAAAGGGATACGTCGAAATCGACCACGCACTGCCGTTCTCGAGAACATGGGACGACTCGTTCAACAACAAGGTCCTGGTCCTGGGATCGGAAAACCAGAACAAGGGAAACCAGACACCGTACGAATACTTCAACGGAAAGGACAACTCGAGAGAATGGCAGGAATTCAAGGCAAGAGTCGAAACATCGAGATTCCCGAGATCGAAGAAGCAGAGAATCCTGCTGCAGAAGTTCGACGAAGACGGATTCAAGGAAAGAAACCTGAACGACACAAGATACGTCAACAGATTCCTGTGCCAGTTCGTCGCAGACAGAATGAGACTGACAGGAAAGGGAAAGAAGAGAGTCTTCGCATCGAACGGACAGATCACAAACCTGCTGAGAGGATTCTGGGGACTGAGAAAGGTCAGAGCAGAAAACGACAGACACCACGCACTGGACGCAGTCGTCGTCGCATGCTCGACAGTCGCAATGCAGCAGAAGATCACAAGATTCGTCAGATACAAGGAAATGAACGCATTCGACGGAAAGACAATCGACAAGGAAACAGGAGAAGTCCTGCACCAGAAGACACACTTCCCGCAGCCGTGGGAATTCTTCGCACAGGAAGTCATGATCAGAGTCTTCGGAAAGCCGGACGGAAAGCCGGAATTCGAAGAAGCAGACACACTGGAAAAGCTGAGAACACTGCTGGCAGAAAAGCTGTCGTCGAGACCGGAAGCAGTCCACGAATACGTCACACCGCTGTTCGTCTCGAGAGCACCGAACAGAAAGATGTCGGGACAGGGACACATGGAAACAGTCAAGTCGGCAAAGAGACTGGACGAAGGAGTCTCGGTCCTGAGAGTCCCGCTGACACAGCTGAAGCTGAAGGACCTGGAAAAGATGGTCAACAGAGAAAGAGAACCGAAGCTGTACGAAGCACTGAAGGCAAGACTGGAAGCACACAAGGACGACCCGGCAAAGGCATTCGCAGAACCGTTCTACAAGTACGACAAGGCAGGAAACAGAACACAGCAGGTCAAGGCAGTCAGAGTCGAACAGGTCCAGAAGACAGGAGTCTGGGTCAGAAACCACAACGGAATCGCAGACAACGCAACAATGGTCAGAGTAGACGTCTTCGAAAAGGGAGACAAGTACTACCTGGTCCCGATCTACTCGTGGCAGGTCGCAAAGGGAATCCTGCCGGACAGAGCAGTCGTCCAGGGAAAGGACGAAGAAGACTGGCAGCTGATCGACGACTCGTTCAACTTCAAGTTCTCGCTGCACCCGAACGACCTGGTCGAAGTCATCACAAAGAAGGCAAGAATGTTCGGATACTTCGCATCGTGCCACAGAGGAACAGGAAACATCAACATCAGAATCCACGACCTGGACCACAAGATCGGAAAGAACGGAATCCTGGAAGGAATCGGAGTCAAGACAGCACTGTCGTTCCAGAAGTACCAGATCGACGAACTGGGAAAGGAAATCAGACCGTGCAGACTGAAGAAGAGACCGCCGGTCAGATCCGGAAAGAGAACAGCAGACGGATCGGAATTCGAATCGCCGAAGAAGAAGAGAAAGGTCGAATGATAGCTAGCTCGAGTCTAGAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATG CGGTGGGCTCTATGGAmino acid MAAFKPNSINYILGLDIGIASVGWAMVEIDEEENPIRLIDLGVRVFERAEV 268sequence of PKTGDSLAMARRLARSVRRLIRRRAHRLLRIRRLLKREGVLQAANFDENGLNeisseria IKSLPNTPWQLRAAALDRKLIPLEWSAVLLHLIKHRGYLSQRKNEGETADKmeningitidis ELGALLKGVAGNAHALQTGDFRIPAELALNKFEKESGHIRNQRSDYSHIFS Cas9RKDLQAELILLFEKQKEFGNPHVSGGLKEGIETLLMTQRPALSGDAVQKMLGHCIFEPAEPKAAKNTYTAERFIWLIKLNNLRILEQGSERPLIDTERAILMDEPYRKSKLIYAQARKLLGLEDTAFFKGLRYGKDNAEASTLMEMKAYHAISRALEKEGLKDKKSPLNLSPELQDEIGTAFSLEKTDEDITGRLKDRIQPEILEALLKHISFDKFVQISLKALRRIVPLMEQGKRYDEACAEIYGDHYGKKNTEEKIYLPPIPADEIRNPVVLRALSQARKVINGVVRRYGSPARIHIETAREVGKSFKDRKEIEKRQEENRKDREKAAAKFREYFPNEVGEPKSKDILKLRLYEQQHGKCLYSGKEINLGRLNEKGYVEIDHALPFSRTWDDSENNKVLVLGSENQNKGNQTPYEYENGKDNSREWQEFKARVETSRFPRSKKQRILLQKFDEDGEKERNLNDTRYVNRFLCQFVADRMRLTGKGKKRVFASNGQITNLLRGFWGLRKVRAENDRHHALDAVVVACSTVAMQQKITREVRYKEMNAFDGKTIDKETGEVLHQKTHFPQPWEFFAQEVMIRVFGKPDGKPEFEEADTLEKLRTLLAEKLSSRPEAVHEYVTPLEVSRAPNRKMSGQGHMETVKSAKRLDEGVSVLRVPLTQLKLKDLEKMVNREREPKLYEALKARLEAHKDDPAKAFAEPFYKYDKAGNRTQQVKAVRVEQVQKTGVWVRNHNGIADNATMVRVDVFEKGDKYYLVPIYSWQVAKGILPDRAVVQGKDEEDWQLIDDSFNEKESLHPNDLVEVITKKARMFGYFASCHRGTGNINIRIHDLDHKIGKNGILEGIGVKTALSFQKYQIDELGKEIRPCRLKKRPPVRSGKRTADGSEFESPKKKRKVE G390 singlemG*mC*mC*GAGUCUGGAGAGCUGCAGUUUUAGAmGmCmUmAmGmAmAmAm 269 guide RNAUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA targetingmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU the rat TTR genetrRNA AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGU 270GGCACCGAGUCGGUGCUUUUUUU Not Used 271 G534 singlemA*mC*mG*CAAAUAUCAGUCCAGCGGUUUUAGAmGmCmUmAmGmAmAmAm 272 guide RNAUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA targetingmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU the rat TTR geneG000395 5′ mG*mC*mA*AUGGUGUAGCGGGUUUUAGAmGmCmUmAmGmAmAmAmUmAmG 273truncated mCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmG inactiveGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sgRNA modified sequenceSV40 NLS PKKKRKV 274 Alternate PKKKRRV 275 SV40 NLS NucleoplasminKRPAATKKAGQAKKKK 276 NLS Exemplary gccRccAUGG 277 Kozak sequenceExemplary gccgccRccAUGG 278 Kozak sequence * = PS linkage; ′m′ = 2T-O-Menucleotide

The following sequence table provides a listing of sequences disclosedherein. It is understood that if a DNA sequence (comprising Ts) isreferenced with respect to an RNA, then Ts should be replaced with Us(which may be modified or unmodified depending on the context), and viceversa.

What is claimed is:
 1. A method of inducing a double-stranded break(DSB) within the TTR gene, comprising delivering a composition to acell, wherein the composition comprises a. a guide RNA comprising aguide sequence selected from SEQ ID NOs: 5-82; b. a guide RNA comprisingat least 17, 18, 19, or 20 contiguous nucleotides of a sequence selectedfrom SEQ ID NOs: 5-82; or c. a guide RNA comprising a guide sequencethat is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90%identical to a sequence selected from SEQ ID NOs: 5-82.
 2. A method ofmodifying the TTR gene comprising delivering a composition to a cell,wherein the composition comprises (i) an RNA-guided DNA binding agent ora nucleic acid encoding an RNA-guided DNA binding agent and (ii) a guideRNA comprising: a. a guide sequence selected from SEQ ID NOs: 5-82; b.at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selectedfrom SEQ ID NOs: 5-82; or c. a guide sequence that is at least 99%, 98%,97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to a sequenceselected from SEQ ID NOs: 5-82.
 3. A method of treating amyloidosisassociated with TTR (ATTR), comprising administering a composition to asubject in need thereof, wherein the composition comprises (i) anRNA-guided DNA binding agent or a nucleic acid encoding an RNA-guidedDNA binding agent and (ii) a guide RNA comprising: a. a guide sequenceselected from SEQ ID NOs: 5-82; b. at least 17, 18, 19, or 20 contiguousnucleotides of a sequence selected from SEQ ID NOs: 5-82; or c. a guidesequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%,or 90% identical to a sequence selected from SEQ ID NOs: 5-82, therebytreating ATTR.
 4. A method of reducing TTR serum concentration,comprising administering a composition to a subject in need thereof,wherein the composition comprises (i) an RNA-guided DNA binding agent ora nucleic acid encoding an RNA-guided DNA binding agent and (ii) a guideRNA comprising: a. a guide sequence selected from SEQ ID NOs: 5-82; b.at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selectedfrom SEQ ID NOs: 5-82; or c. a guide sequence that is at least 99%, 98%,97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to a sequenceselected from SEQ ID NOs: 5-82, thereby reducing TTR serumconcentration.
 5. A method for reducing or preventing the accumulationof amyloids or amyloid fibrils comprising TTR in a subject, comprisingadministering a composition to a subject in need thereof, wherein thecomposition comprises (i) an RNA-guided DNA binding agent or a nucleicacid encoding an RNA-guided DNA binding agent and (ii) a guide RNAcomprising: a. a guide sequence selected from SEQ ID NOs: 5-82; b. atleast 17, 18, 19, or 20 contiguous nucleotides of a sequence selectedfrom SEQ ID NOs: 5-82; or c. a guide sequence that is at least 99%, 98%,97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to a sequenceselected from SEQ ID NOs: 5-82, thereby reducing accumulation ofamyloids or amyloid fibrils.
 6. A composition comprising a guide RNAcomprising: a. a guide sequence selected from SEQ ID NOs: 5-82; b. atleast 17, 18, 19, or 20 contiguous nucleotides of a sequence selectedfrom SEQ ID NOs: 5-82; or c. a guide sequence that is at least 99%, 98%,97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to a sequenceselected from SEQ ID NOs: 5-82.
 7. A composition comprising a vectorencoding a guide RNA, wherein the guide RNA comprises: a. a guidesequence selected from SEQ ID NOs: 5-82; b. at least 17, 18, 19, or 20contiguous nucleotides of a sequence selected from SEQ ID NOs: 5-82; orc. a guide sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%,92%, 91%, or 90% identical to a sequence selected from SEQ ID NOs: 5-82.8. The composition of claim 6 or 7, for use in inducing adouble-stranded break (DSB) within the TTR gene in a cell or subject. 9.The composition of claim 6 or 7, for use in modifying the TTR gene in acell or subject.
 10. The composition of claim 6 or 7, for use intreating amyloidosis associated with TTR (ATTR) in a subject.
 11. Thecomposition of claim 6 or 7, for use in reducing TTR serum concentrationin a subject.
 12. The composition of claim 6 or 7, for use in reducingor preventing the accumulation of amyloids or amyloid fibrils in asubject.
 13. The method of any one of claims 1-5 or the composition foruse of any one of claims 8-12, wherein the composition reduces serum TTRlevels.
 14. The method or composition for use of claim 13, wherein theserum TTR levels are reduced by at least 50% as compared to serum TTRlevels before administration of the composition.
 15. The method orcomposition for use of claim 13, wherein the serum TTR levels arereduced by 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, 95-98%, 98-99%, or99-100% as compared to serum TTR levels before administration of thecomposition.
 16. The method or composition for use of any one of claim1-5 or 8-15, wherein the composition results in editing of the TTR gene.17. The method or composition for use of claim 16, wherein the editingis calculated as a percentage of the population that is edited (percentediting).
 18. The method or composition for use of claim 17, wherein thepercent editing is between 30 and 99% of the population.
 19. The methodor composition for use of claim 17, wherein the percent editing isbetween 30 and 35%, 35 and 40%, 40 and 45%, 45 and 50%, 50 and 55%, 55and 60%, 60 and 65%, 65 and 70%, 70 and 75%, 75 and 80%, 80 and 85%, 85and 90%, 90 and 95%, or 95 and 99% of the population.
 20. The method ofany one of claims 1-5 or the composition for use of any one of claims8-19, wherein the composition reduces amyloid deposition in at least onetissue.
 21. The method or composition for use of claim 20, wherein theat least one tissue comprises one or more of stomach, colon, sciaticnerve, or dorsal root ganglion.
 22. The method or composition for use ofclaim 20 or 21, wherein amyloid deposition is measured 8 weeks afteradministration of the composition.
 23. The method or composition for useof any one of claims 20-22, wherein amyloid deposition is compared to anegative control or a level measured before administration of thecomposition.
 24. The method or composition for use of any one of claims20-23, wherein amyloid deposition is measured in a biopsy sample and/orby immunostaining.
 25. The method or composition for use of any one ofclaims 20-24, wherein amyloid deposition is reduced by between 30 and35%, 35 and 40%, 40 and 45%, 45 and 50%, 50 and 55%, 55 and 60%, 60 and65%, 65 and 70%, 70 and 75%, 75 and 80%, 80 and 85%, 85 and 90%, 90 and95%, or 95 and 99% of the amyloid deposition seen in a negative control.26. The method or composition for use of any one of claims 20-25,wherein amyloid deposition is reduced by between 30 and 35%, 35 and 40%,40 and 45%, 45 and 50%, 50 and 55%, 55 and 60%, 60 and 65%, 65 and 70%,70 and 75%, 75 and 80%, 80 and 85%, 85 and 90%, 90 and 95%, or 95 and99% of the amyloid deposition seen before administration of thecomposition.
 27. The method or composition for use of any one of claim1-5 or 8-26, wherein the composition is administered or delivered atleast two times.
 28. The method or composition for use of claim 27,wherein the composition is administered or delivered at least threetimes.
 29. The method or composition for use of claim 27, wherein thecomposition is administered or delivered at least four times.
 30. Themethod or composition for use of claim 27, wherein the composition isadministered or delivered up to five, six, seven, eight, nine, or tentimes.
 31. The method or composition for use of any one of claims 27-30,wherein the administration or delivery occurs at an interval of 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days.
 32. The method orcomposition for use of any one of claims 27-30, wherein theadministration or delivery occurs at an interval of 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, or 15 weeks.
 33. The method or composition foruse of any one of claims 27-30, wherein the administration or deliveryoccurs at an interval of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,or 15 months.
 34. The method or composition of any one of the precedingclaims, wherein the guide sequence is selected from SEQ ID NOs: 5-82.35. The method or composition of any one of the preceding claims,wherein the guide RNA is at least partially complementary to a targetsequence present in the human TTR gene.
 36. The method or composition ofclaim 35, wherein the target sequence is in exon 1, 2, 3, or 4 of thehuman TTR gene.
 37. The method or composition of claim 35, wherein thetarget sequence is in exon 1 of the human TTR gene.
 38. The method orcomposition of claim 35, wherein the target sequence is in exon 2 of thehuman TTR gene.
 39. The method or composition of claim 35, wherein thetarget sequence is in exon 3 of the human TTR gene.
 40. The method orcomposition of claim 35, wherein the target sequence is in exon 4 of thehuman TTR gene.
 41. The method or composition of any one of claims 1-40,wherein the guide sequence is complementary to a target sequence in thepositive strand of TTR.
 42. The method or composition of any one ofclaims 1-40, wherein the guide sequence is complementary to a targetsequence in the negative strand of TTR.
 43. The method or composition ofany one of claims 1-40, wherein the first guide sequence iscomplementary to a first target sequence in the positive strand of theTTR gene, and wherein the composition further comprises a second guidesequence that is complementary to a second target sequence in thenegative strand of the TTR gene.
 44. The method or composition of anyone of the preceding claims, wherein the guide RNA comprises a crRNAthat comprises the guide sequence and further comprises a nucleotidesequence of SEQ ID NO: 126, wherein the nucleotides of SEQ ID NO: 126follow the guide sequence at its 3′ end.
 45. The method or compositionof any one of the preceding claims, wherein the guide RNA is a dualguide (dgRNA).
 46. The method or composition of claim 45, wherein thedual guide RNA comprises a crRNA comprising a nucleotide sequence of SEQID NO: 126, wherein the nucleotides of SEQ ID NO: 126 follow the guidesequence at its 3′ end, and a trRNA.
 47. The method or composition ofany one of claims 1-43, wherein the guide RNA is a single guide (sgRNA).48. The method or composition of claim 47, wherein the sgRNA comprises aguide sequence that has the pattern of SEQ ID NO:
 3. 49. The method orcomposition of claim 47, wherein the sgRNA comprises the sequence of SEQID NO:
 3. 50. The method or composition of claim 48 or 49, wherein eachN in SEQ ID NO: 3 is any natural or non-natural nucleotide, wherein theN's form the guide sequence, and the guide sequence targets Cas9 to theTTR gene.
 51. The method or composition of any one of claims 47-50,wherein the sgRNA comprises any one of the guide sequences of SEQ IDNOs: 5-82 and the nucleotides of SEQ ID NO:
 126. 52. The method orcomposition of any one of claims 47-51, wherein the sgRNA comprises aguide sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%,91%, or 90% identical to a sequence selected from SEQ ID Nos: 87-124.53. The method or composition of claim 47, wherein the sgRNA comprises asequence selected from SEQ ID Nos: 87-124.
 54. The method or compositionof any one of the preceding claims, wherein the guide RNA comprises atleast one modification.
 55. The method or composition of claim 54,wherein the at least one modification includes a 2′-O-methyl (2′-O-Me)modified nucleotide.
 56. The method or composition of claim 54 or 55,wherein the at least one modification includes a phosphorothioate (PS)bond between nucleotides.
 57. The method or composition of any one ofclaims 54-56, wherein the at least one modification includes a 2′-fluoro(2′-F) modified nucleotide.
 58. The method or composition of any one ofclaims 54-57, wherein the at least one modification includes amodification at one or more of the first five nucleotides at the 5′ end.59. The method or composition of any one of claims 54-58, wherein the atleast one modification includes a modification at one or more of thelast five nucleotides at the 3′ end.
 60. The method or composition ofany one of claims 54-59, wherein the at least one modification includesPS bonds between the first four nucleotides.
 61. The method orcomposition of any one of claims 54-60, wherein the at least onemodification includes PS bonds between the last four nucleotides. 62.The method or composition of any one of claims 54-61, wherein the atleast one modification includes 2′-O-Me modified nucleotides at thefirst three nucleotides at the 5′ end.
 63. The method or composition ofany one of claims 54-62, wherein the at least one modification includes2′-O-Me modified nucleotides at the last three nucleotides at the 3′end.
 64. The method or composition of any one of claims 54-63, whereinthe guide RNA comprises the modified nucleotides of SEQ ID NO:
 3. 65.The method or composition of any one of claims 1-64, wherein thecomposition further comprises a pharmaceutically acceptable excipient.66. The method or composition of any one of claims 1-65, wherein theguide RNA is associated with a lipid nanoparticle (LNP).
 67. The methodor composition of claim 66, wherein the LNP comprises a CCD lipid. 68.The method or composition of claim 67, wherein the CCD lipid is Lipid Aor Lipid B.
 69. The method or composition of claim 66-68, wherein theLNP comprises a neutral lipid.
 70. The method or composition of claim69, wherein the neutral lipid is DSPC
 71. The method or composition ofany one of claims 66-70, wherein the LNP comprises a helper lipid. 72.The method or composition of claim 71, wherein the helper lipid ischolesterol.
 73. The method or composition of any one of claims 66-72,wherein the LNP comprises a stealth lipid.
 74. The method or compositionof claim 73, wherein the stealth lipid is PEG2k-DMG.
 75. The method orcomposition of any one of the preceding claims, wherein the compositionfurther comprises an RNA-guided DNA binding agent.
 76. The method orcomposition of any one of the preceding claims, wherein the compositionfurther comprises an mRNA that encodes an RNA-guided DNA binding agent.77. The method or composition of claim 75 or 76, wherein the RNA-guidedDNA binding agent is a Cas cleavase.
 78. The method or composition ofclaim 77, wherein the RNA-guided DNA binding agent is Cas9.
 79. Themethod or composition of any one of claims 75-78, wherein the RNA-guidedDNA binding agent is modified.
 80. The method or composition of any oneof claims 75-79, wherein the RNA-guided DNA binding agent is a nickase.81. The method or composition of claim 79 or 80, wherein the modifiedRNA-guided DNA binding agent comprises a nuclear localization signal(NLS).
 82. The method or composition of any one of claims 75-81, whereinthe RNA-guided DNA binding agent is a Cas from a Type-II CRISPR/Cassystem.
 83. The method or composition of any one of the precedingclaims, wherein the composition is a pharmaceutical formulation andfurther comprises a pharmaceutically acceptable carrier.
 84. The methodor composition for use of any one of claim 1-5 or 8-83, wherein thecomposition reduces or prevents amyloids or amyloid fibrils comprisingTTR.
 85. The method or composition for use of claim 84, wherein theamyloids or amyloid fibrils are in the nerves, heart, orgastrointestinal track.
 86. The method or composition for use of any oneof claim 1-5 or 8-83, wherein non-homologous ending joining (NHEJ) leadsto a mutation during repair of a DSB in the TTR gene.
 87. The method orcomposition for use of claim 86, wherein NHEJ leads to a deletion orinsertion of a nucleotide(s) during repair of a DSB in the TTR gene. 88.The method or composition for use of claim 87, wherein the deletion orinsertion of a nucleotide(s) induces a frame shift or nonsense mutationin the TTR gene.
 89. The method or composition for use of claim 87,wherein a frame shift or nonsense mutation is induced in the TTR gene ofat least 50% of liver cells.
 90. The method or composition for use ofclaim 89, wherein a frame shift or nonsense mutation is induced in theTTR gene of 50%-60%, 60%-70%, 70% or 80%, 80%-90%, 90-95%, 95%-99%, or99%-100% of liver cells.
 91. The method or composition for use of anyone of claims 87-90, wherein a deletion or insertion of a nucleotide(s)occurs in the TTR gene at least 50-fold or more than in off-targetsites.
 92. The method or composition for use of claim 91, wherein thedeletion or insertion of a nucleotide(s) occurs in the TTR gene 50-foldto 150-fold, 150-fold to 500-fold, 500-fold to 1500-fold, 1500-fold to5000-fold, 5000-fold to 15000-fold, 15000-fold to 30000-fold, or30000-fold to 60000-fold more than in off-target sites.
 93. The methodor composition for use of any one of claims 87-92, wherein the deletionor insertion of a nucleotide(s) occurs at less than or equal to 3, 2, 1,or 0 off-target site(s) in primary human hepatocytes, optionally whereinthe off-target site(s) does (do) not occur in a protein coding region inthe genome of the primary human hepatocytes.
 94. The method orcomposition for use of claim 93, wherein the deletion or insertion of anucleotide(s) occurs at a number of off-target sites in primary humanhepatocytes that is less than the number of off-target sites at which adeletion or insertion of a nucleotide(s) occurs in Cas9-overexpressingcells, optionally wherein the off-target site(s) does (do) not occur ina protein coding region in the genome of the primary human hepatocytes.95. The method or composition for use of claim 94, wherein theCas9-overexpressing cells are HEK293 cells stably expressing Cas9. 96.The method or composition for use of any one of claims 93-95, whereinthe number of off-target sites in primary human hepatocytes isdetermined by analyzing genomic DNA from primary human hepatocytestransfected in vitro with Cas9 mRNA and the guide RNA, optionallywherein the off-target site(s) does (do) not occur in a protein codingregion in the genome of the primary human hepatocytes.
 97. The method orcomposition for use of any one of claims 93-95, wherein the number ofoff-target sites in primary human hepatocytes is determined by anoligonucleotide insertion assay comprising analyzing genomic DNA fromprimary human hepatocytes transfected in vitro with Cas9 mRNA, the guideRNA, and a donor oligonucleotide, optionally wherein the off-targetsite(s) does (do) not occur in a protein coding region in the genome ofthe primary human hepatocytes.
 98. The method or composition of any oneof claim 1-43 or 47-97, wherein the sequence of the guide RNA is: a) SEQID NO: 92 or 104; b) SEQ ID NO: 87, 89, 96, or 113; c) SEQ ID NO: 100,102, 106, 111, or 112; or d) SEQ ID NO: 88, 90, 91, 93, 94, 95, 97, 101,103, 108, or 109, optionally wherein the guide RNA does not produceindels at off-target site(s) that occur in a protein coding region inthe genome of primary human hepatocytes.
 99. The method or compositionfor use of any one of claim 1-5 or 8-98, wherein administering thecomposition reduces levels of TTR in the subject.
 100. The method orcomposition for use of claim 99, wherein the levels of TTR are reducedby at least 50%.
 101. The method or composition for use of claim 100,wherein the levels of TTR are reduced by 50%-60%, 60%-70%, 70% or 80%,80%-90%, 90-95%, 95%-99%, or 99%-100%.
 102. The method or compositionfor use of claim 100 or 101, wherein the levels of TTR are measured inserum, plasma, blood, cerebral spinal fluid, or sputum.
 103. The methodor composition for use of claim 100 or 101, wherein the levels of TTRare measured in liver, choroid plexus, and/or retina.
 104. The method orcomposition for use of any one of claims 99-103, wherein the levels ofTTR are measured via enzyme-linked immunosorbent assay (ELISA).
 105. Themethod or composition for use of any one of claim 1-5 or 8-104, whereinthe subject has ATTR.
 106. The method or composition for use of any oneof claim 1-5 or 8-105, wherein the subject is human.
 107. The method orcomposition for use of claim 105 or 106, wherein the subject has ATTRwt.108. The method or composition for use of claim 105 or 106, wherein thesubject has hereditary ATTR.
 109. The method or composition for use ofany one of claim 1-5, 8-106, or 108, wherein the subject has a familyhistory of ATTR.
 110. The method or composition for use of any one ofclaim 1-5, 8-106, or 108-109, wherein the subject has familial amyloidpolyneuropathy.
 111. The method or composition for use of any one ofclaim 1-5 or 8-110, wherein the subject has only or predominantly nervesymptoms of ATTR.
 112. The method or composition for use of any one ofclaim 1-5 or 8-110, wherein the subject has familial amyloidcardiomyopathy.
 113. The method or composition for use of any one ofclaim 1-5, 8-109, or 112, wherein the subject has only or predominantlycardiac symptoms of ATTR.
 114. The method or composition for use of anyone of claim 1-5 or 8-113, wherein the subject expresses TTR having aV30 mutation.
 115. The method or composition for use of claim 114,wherein the V30 mutation is V30A, V30G, V30L, or V30M.
 116. The methodor composition for use of claim any one of claim 1-5 or 8-113, whereinthe subject expresses TTR having a T60 mutation.
 117. The method orcomposition for use of claim 116, wherein the T60 mutation is T60A. 118.The method or composition for use of claim any one of claim 1-5 or8-113, wherein the subject expresses TTR having a V122 mutation. 119.The method or composition for use of claim 118, wherein the V122mutation is V122A, V122I, or V122(−).
 120. The method or composition foruse of any one of claim 1-5 or 8-119, wherein the subject expresseswild-type TTR.
 121. The method or composition for use of any one ofclaim 1-5, 8-107, or 120, wherein the subject does not express TTRhaving a V30, T60, or V122 mutation.
 122. The method or composition foruse of any one of claim 1-5, 8-107, or 120-121, wherein the subject doesnot express TTR having a pathological mutation.
 123. The method orcomposition for use of claim 121, wherein the subject is homozygous forwild-type TTR.
 124. The method or composition for use of any one ofclaim 1-5 or 8-123, wherein after administration the subject has animprovement, stabilization, or slowing of change in symptoms ofsensorimotor neuropathy.
 125. The method or composition for use of claim124, wherein the improvement, stabilization, or slowing of change insensory neuropathy is measured using electromyogram, nerve conductiontests, or patient-reported outcomes.
 126. The method or composition foruse of any one of claim 1-5 or 8-125, wherein the subject has animprovement, stabilization, or slowing of change in symptoms ofcongestive heart failure.
 127. The method or composition for use ofclaim 126, wherein the improvement, stabilization, or slowing of changein congestive heart failure is measured using cardiac biomarker tests,lung function tests, chest x-rays, or electrocardiography.
 128. Themethod or composition for use of any one of claim 1-5 or 8-127, whereinthe composition or pharmaceutical formulation is administered via aviral vector.
 129. The method or composition for use of any one of claim1-5 or 8-127, wherein the composition or pharmaceutical formulation isadministered via lipid nanoparticles.
 130. The method or composition foruse of any one of claim 1-5 or 8-129, wherein the subject is tested forspecific mutations in the TTR gene before administering the compositionor formulation.
 131. The method or composition of any one of claims1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:5.
 132. The method or composition of any one of claims 1-130, whereinthe sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:
 6. 133. Themethod or composition of any one of claims 1-130, wherein the sequenceselected from SEQ ID NOs: 5-82 is SEQ ID NO:
 7. 134. The method orcomposition of any one of claims 1-130, wherein the sequence selectedfrom SEQ ID NOs: 5-82 is SEQ ID NO:
 8. 135. The method or composition ofany one of claims 1-130, wherein the sequence selected from SEQ ID NOs:5-82 is SEQ ID NO:
 9. 136. The method or composition of any one ofclaims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQID NO:
 10. 137. The method or composition of any one of claims 1-130,wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 11.138. The method or composition of any one of claims 1-130, wherein thesequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:
 12. 139. Themethod or composition of any one of claims 1-130, wherein the sequenceselected from SEQ ID NOs: 5-82 is SEQ ID NO:
 13. 140. The method orcomposition of any one of claims 1-130, wherein the sequence selectedfrom SEQ ID NOs: 5-82 is SEQ ID NO:
 14. 141. The method or compositionof any one of claims 1-130, wherein the sequence selected from SEQ IDNOs: 5-82 is SEQ ID NO:
 15. 142. The method or composition of any one ofclaims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQID NO:
 16. 143. The method or composition of any one of claims 1-130,wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 17.144. The method or composition of any one of claims 1-130, wherein thesequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:
 18. 145. Themethod or composition of any one of claims 1-130, wherein the sequenceselected from SEQ ID NOs: 5-82 is SEQ ID NO:
 19. 146. The method orcomposition of any one of claims 1-130, wherein the sequence selectedfrom SEQ ID NOs: 5-82 is SEQ ID NO:
 20. 147. The method or compositionof any one of claims 1-130, wherein the sequence selected from SEQ IDNOs: 5-82 is SEQ ID NO:
 21. 148. The method or composition of any one ofclaims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQID NO:
 22. 149. The method or composition of any one of claims 1-130,wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 23.150. The method or composition of any one of claims 1-130, wherein thesequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:
 24. 151. Themethod or composition of any one of claims 1-130, wherein the sequenceselected from SEQ ID NOs: 5-82 is SEQ ID NO:
 25. 152. The method orcomposition of any one of claims 1-130, wherein the sequence selectedfrom SEQ ID NOs: 5-82 is SEQ ID NO:
 26. 153. The method or compositionof any one of claims 1-130, wherein the sequence selected from SEQ IDNOs: 5-82 is SEQ ID NO:
 27. 154. The method or composition of any one ofclaims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQID NO:
 28. 155. The method or composition of any one of claims 1-130,wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 29.156. The method or composition of any one of claims 1-130, wherein thesequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:
 30. 157. Themethod or composition of any one of claims 1-130, wherein the sequenceselected from SEQ ID NOs: 5-82 is SEQ ID NO:
 31. 158. The method orcomposition of any one of claims 1-130, wherein the sequence selectedfrom SEQ ID NOs: 5-82 is SEQ ID NO:
 32. 159. The method or compositionof any one of claims 1-130, wherein the sequence selected from SEQ IDNOs: 5-82 is SEQ ID NO:
 33. 160. The method or composition of any one ofclaims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQID NO:
 34. 161. The method or composition of any one of claims 1-130,wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 35.162. The method or composition of any one of claims 1-130, wherein thesequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:
 36. 163. Themethod or composition of any one of claims 1-130, wherein the sequenceselected from SEQ ID NOs: 5-82 is SEQ ID NO:
 37. 164. The method orcomposition of any one of claims 1-130, wherein the sequence selectedfrom SEQ ID NOs: 5-82 is SEQ ID NO:
 38. 165. The method or compositionof any one of claims 1-130, wherein the sequence selected from SEQ IDNOs: 5-82 is SEQ ID NO:
 39. 166. The method or composition of any one ofclaims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQID NO:
 40. 167. The method or composition of any one of claims 1-130,wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 41.168. The method or composition of any one of claims 1-130, wherein thesequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:
 42. 169. Themethod or composition of any one of claims 1-130, wherein the sequenceselected from SEQ ID NOs: 5-82 is SEQ ID NO:
 43. 170. The method orcomposition of any one of claims 1-130, wherein the sequence selectedfrom SEQ ID NOs: 5-82 is SEQ ID NO:
 44. 171. The method or compositionof any one of claims 1-130, wherein the sequence selected from SEQ IDNOs: 5-82 is SEQ ID NO:
 45. 172. The method or composition of any one ofclaims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQID NO:
 46. 173. The method or composition of any one of claims 1-130,wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 47.174. The method or composition of any one of claims 1-130, wherein thesequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:
 48. 175. Themethod or composition of any one of claims 1-130, wherein the sequenceselected from SEQ ID NOs: 5-82 is SEQ ID NO:
 49. 176. The method orcomposition of any one of claims 1-130, wherein the sequence selectedfrom SEQ ID NOs: 5-82 is SEQ ID NO:
 50. 177. The method or compositionof any one of claims 1-130, wherein the sequence selected from SEQ IDNOs: 5-82 is SEQ ID NO:
 51. 178. The method or composition of any one ofclaims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQID NO:
 52. 179. The method or composition of any one of claims 1-130,wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 53.180. The method or composition of any one of claims 1-130, wherein thesequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:
 54. 181. Themethod or composition of any one of claims 1-130, wherein the sequenceselected from SEQ ID NOs: 5-82 is SEQ ID NO:
 55. 182. The method orcomposition of any one of claims 1-130, wherein the sequence selectedfrom SEQ ID NOs: 5-82 is SEQ ID NO:
 56. 183. The method or compositionof any one of claims 1-130, wherein the sequence selected from SEQ IDNOs: 5-82 is SEQ ID NO:
 57. 184. The method or composition of any one ofclaims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQID NO:
 58. 185. The method or composition of any one of claims 1-130,wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 59.186. The method or composition of any one of claims 1-130, wherein thesequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:
 60. 187. Themethod or composition of any one of claims 1-130, wherein the sequenceselected from SEQ ID NOs: 5-82 is SEQ ID NO:
 61. 188. The method orcomposition of any one of claims 1-130, wherein the sequence selectedfrom SEQ ID NOs: 5-82 is SEQ ID NO:
 62. 189. The method or compositionof any one of claims 1-130, wherein the sequence selected from SEQ IDNOs: 5-82 is SEQ ID NO:
 63. 190. The method or composition of any one ofclaims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQID NO:
 64. 191. The method or composition of any one of claims 1-130,wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 65.192. The method or composition of any one of claims 1-130, wherein thesequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:
 66. 193. Themethod or composition of any one of claims 1-130, wherein the sequenceselected from SEQ ID NOs: 5-82 is SEQ ID NO:
 67. 194. The method orcomposition of any one of claims 1-130, wherein the sequence selectedfrom SEQ ID NOs: 5-82 is SEQ ID NO:
 68. 195. The method or compositionof any one of claims 1-130, wherein the sequence selected from SEQ IDNOs: 5-82 is SEQ ID NO:
 69. 196. The method or composition of any one ofclaims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQID NO:
 70. 197. The method or composition of any one of claims 1-130,wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 71.198. The method or composition of any one of claims 1-130, wherein thesequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:
 72. 199. Themethod or composition of any one of claims 1-130, wherein the sequenceselected from SEQ ID NOs: 5-82 is SEQ ID NO:
 73. 200. The method orcomposition of any one of claims 1-130, wherein the sequence selectedfrom SEQ ID NOs: 5-82 is SEQ ID NO:
 74. 201. The method or compositionof any one of claims 1-130, wherein the sequence selected from SEQ IDNOs: 5-82 is SEQ ID NO:
 75. 202. The method or composition of any one ofclaims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQID NO:
 76. 203. The method or composition of any one of claims 1-130,wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 77.204. The method or composition of any one of claims 1-130, wherein thesequence selected from SEQ ID NOs: 5-82 is SEQ ID NO:
 78. 205. Themethod or composition of any one of claims 1-130, wherein the sequenceselected from SEQ ID NOs: 5-82 is SEQ ID NO:
 79. 206. The method orcomposition of any one of claims 1-130, wherein the sequence selectedfrom SEQ ID NOs: 5-82 is SEQ ID NO:
 80. 207. The method or compositionof any one of claims 1-130, wherein the sequence selected from SEQ IDNOs: 5-82 is SEQ ID NO:
 81. 208. The method or composition of any one ofclaims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQID NO:
 82. 209. Use of a composition or formulation of any of claims6-208 for the preparation of a medicament for treating a human subjecthaving ATTR.